Patent application title: THERAPEUTIC AGENTS FOR ALZHEIMER'S DISEASE AND CANCER
Takeshi Iwatsubo (Tokyo, JP)
Tatsuhiko Kodama (Tokyo, JP)
Takao Hamakuob (Tokyo, JP)
Taisuke Tomita (Tokyo, JP)
Ikuo Hayashi (Tokyo, JP)
Yasuomi Urano (Tokyo, JP)
Hiroko Iwanari (Tochigi, JP)
Masao Ohkuchi (Saitama, JP)
THE UNIVERSITY OF TOKYO
Perseus Proteomics Inc.
IPC8 Class: AA61K39395FI
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds hormone or other secreted growth regulatory factor, differentiation factor, or intercellular mediator (e.g., cytokine, vascular permeability factor, etc.); or binds serum protein, plasma protein, fibrin, or enzyme
Publication date: 2009-09-03
Patent application number: 20090220524
To provide a therapeutic drug for Alzheimer's disease and/or a cancer.
The therapeutic drug for Alzheimer's disease and/or a cancer contains an
anti-nicastrin antibody, a derivative of the antibody, or a fragment of
the antibody or the derivative.
1. A therapeutic drug for Alzheimer's disease and/or a cancer containing
an anti-nicastrin antibody, a derivative of the antibody, or a fragment
of the antibody or the derivative.
2. The therapeutic drug as described in claim 1, wherein the cancer is a nicastrin-expressing cancer.
3. The therapeutic drug for a cancer as described in claim 1, wherein the cancer is a γ-secretase-dependent cancer.
4. The therapeutic drug as described in claim 1, wherein the cancer is lung cancer or T-cell acute lymphoblastic leukemia.
5. A screening method for selecting an antibody which inhibits γ-secretase activity, characterized by comprising reacting nicastrin with a γ-secretase substrate in the presence of a test antibody.
6. Use of an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative for producing a therapeutic drug for Alzheimer's disease and/or a cancer.
7. Use as described in claim 6, wherein the cancer is a nicastrin-expressing cancer.
8. Use as described in claim 6, wherein the cancer is a γ-secretase-dependent cancer.
9. Use as described in claim 6, wherein the cancer is lung cancer or T-cell acute lymphoblastic leukemia.
10. A method for treatment of Alzheimer's disease and/or a cancer, comprising administering an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative to a subject in need thereof.
11. The method as described in claim 10, wherein the cancer is a nicastrin-expressing cancer.
12. The method as described in claim 10, wherein the cancer is a γ-secretase-dependent cancer.
13. The method as described in claim 10, wherein the cancer is lung cancer or T-cell acute lymphoblastic leukemia.
The present invention relates to a therapeutic drug for Alzheimer's disease and/or a cancer, the drug containing an anti-nicastrin antibody.
In Japan, the three most common death-causing diseases are cancer (30.3%), cardiac disease (15.3%), and cerebrovascular disease (15.2%). As the population ages, the percentage of patients with such diseases increases, which greatly affects medical costs required for treatment or nursing care. In recent years, a nursing-care insurance system for cerebrovascular disease patients has been established as a national policy.
In Japan, the number of deaths from cancer was 320,315 (i.e., 253.9 per 100,000) in 2004. In Japan, in 2003, lung cancer (22.3%) was ranked first among cancer deaths in men, followed by gastric cancer (17.2%) and liver cancer (12.5%), whereas colon cancer (14.6%) was ranked first among cancer deaths in women, followed by gastric cancer (14.2%) and lung cancer (12.3%). According to a report by the National Cancer Center in Japan, regarding five-year survival rates for major types of cancer, the five-year survival rate of pancreatic cancer patients is the lowest (only a few percent), followed by that of patients with gallbladder cancer, lung and bronchial cancer, liver cancer, esophageal cancer, etc. Ohno, Nakamura, et al. have estimated that the number of new cases of male cancers will be 501,000 in 2020 (major sites of cancer: lung, prostate gland, stomach, colon, liver, etc.), whereas the number of new cases of female cancers will be 337,000 in 2020 (major sites of cancer: breast, colon, stomach, lung, uterus, rectum, liver, etc.). Thus, cancer is predicted to become a major death-causing disease in future (as is the case at present), and development of a therapy for cancer is essential.
Cancer therapy has changed with the times. Recently, in addition to hitherto performed surgery, drug therapy, and radiotherapy, endoscopic resection of cancer tissue has been carried out, and chemotherapy for outpatients has been performed more and more. However, about 40% of cancer cases are treated through surgery at present, and radiotherapy or chemotherapy is less effective for some cancers (e.g., pancreatic cancer). In some cancer cases, chemotherapy can reduce a size of cancer tissue, but encounters difficulty in completely curing the disease. In many refractory cancer cases, adverse reactions to an anticancer agent (i.e., side effects thereof) are more pronounced than the effects of the drug.
Cerebrovascular diseases are classified into a cognitive disorder, which is caused by vascular disorder, and Alzheimer's disease, which is a neurodegenerative disease. In Japan, a number of patients with dementia caused by Alzheimer's disease (AD) has increased with adoption of Europeanized and Americanized meals and aging of the population.
AD is a neurodegenerative disease which develops various intellectual dysfunctions (including memory impairment) due to degeneration or loss of cerebral cortical neurons. An AD brain is characterized by accumulation of an abnormal protein called "β-amyloid," which is closely related to loss of neurons (Non-Patent Document 1).
β-Amyloid is accumulated in an AD brain in the pathological form of senile plaque or vascular amyloid. From the biochemical viewpoint, β-amyloid is formed of Aβ peptide including 40 to 42 amino acid residues. Aβ is produced from APP (amyloid precursor protein) through two-step cleavage and is secreted extracellularly. In the second step, a C-terminal fragment of APP is cleaved at an intramembrane site by the protease activity of the enzyme γ-secretase, and the thus-formed Aβ is released extracellularly. Cleavage of the C-terminal fragment of APP occurs at different sites; i.e., at position 40 (90%) and at position 42 (10%) (Non-Patent Document 2). Aβ42 is more highly aggregated in the form of β-amyloid and is preferentially accumulated in an AD brain from an early stage (Non-Patent Document 3).
As has been shown, presenilin (PS) protein, which is an expression product of a major pathogenic gene of familial AD, corresponds to a catalytic subunit of γ-secretase, which is a membrane-associated aspartic protease (Non-Patent Documents 4 and 5).
γ-Secretase has been shown to be involved not only in AD but also in Notch signaling (Non-Patent Document 6). As has been known, a γ-secretase inhibitor (i.e., a low-molecular-weight compound) induces apoptosis in Kaposi's sarcoma (Non-Patent Document 7) or inhibits survival of T-ALL cells (Non-Patent Document 8). However, it has been reported that a γ-secretase inhibitor may promote malignant transformation (Non-Patent Document 9). Thus, inhibition of Notch signaling does not necessarily induce cell death in all cancers, and in the future studies will be carried out to determine whether or not a γ-secretase inhibitor can be used as a therapeutic drug for cancer.
Under such circumstances, γ-secretase has been considered important as a therapeutic target for AD or cancer, but a cancer therapeutic drug based on γ-secretase has not successfully been developed for, for example, the following reason. Since γ-secretase is a complex formed of a plurality of membrane proteins and exhibits protease activity in the membrane, difficulty is encountered in reconstituting γ-secretase while maintaining protease activity, and drug screening is not properly carried out by use of γ-secretase.
As has been known, human active γ-secretase complex is a large membrane protein complex having a molecular weight of 250 to 500 kDa or more and including the following four proteins: presenilin, nicastrin (NCT), APH-1, and PEN-2. That is, nicastrin is a constituent molecule of γ-secretase. Many attempts have been made to search for γ-secretase activity inhibitors by use of low-molecular-weight compounds, but no report has been provided to show a result of an experiment by use of an anti-nicastrin antibody for development of a γ-secretase activity inhibitor or a therapy for AD and/or cancer. Although there are many AD and cancer patients, a good drug for a treatment of the diseases has not yet been provided. Development of a therapeutic drug for AD or cancer could reduce burden of nursing care as a matter of course, along with medical costs.
Non-Patent Document 1: Selkoe D J., Physiol. Rev. 2001, 81 (2): 741-766, Alzheimer's disease: genes, proteins, and therapyNon-Patent Document 2: Suzuki N., et al. Science 264: 1336, 1994
Non-Patent Document 3: Iwatsubo T., Odaka A., Suzuki N., Mizusawa H., Nukina N., Ihara Y., Neuron. 1994, 13 (1): 45-53
Non-Patent Document 4: Wolfe M S., Xia W., Ostaszewski B L., Diehl T S., Kimberly W T., Selkoe D J. (1999), Nature 398 (6727): 513-517
Non-Patent Document 5: Li Y M., Xu M., Lai M T., Huang Q., Castro J L., DiMuzio Mower J., Harrison T., Lellis C., Nadin A., Neduvelil J G., Register R B., Sardana M K., Shearman M S., Smith A L., Shi X P., Yin K C., Shafer J A., Gardell S J. (2000), Nature 2000 Jun. 8, 405 (6787): 689-94Non-Patent Document 6: J. Biol. Chem. 2001 Aug. 10; 276 (32): 30018-30023
Non-Patent Document 7: Oncogene. 2005 Sep. 22; 24 (42): 6333-6344
Non-Patent Document 8: Mol. Cell. Biol. 2003 January; 23 (2): 655-664Non-Patent Document 9: Br. J. Cancer. 2005 Sep. 19; 93 (6): 709-718
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
The present invention provides a new therapeutic drug for AD or a cancer. The present invention also provides a screening method for selecting such a therapeutic drug.
Means for Solving the Problems
The present inventors have succeeded in expressing active γ-secretase by use of budding baculovirus (see WO 2005/038023). The present inventors have screened anti-nicastrin antibodies on a basis of active γ-secretase activity by use of budding baculovirus, and as a result have found that an excellent anti-nicastrin antibody is useful as a therapeutic drug for AD and/or a cancer, since the anti-nicastrin antibody exhibits γ-secretase-neutralizing activity and inhibits proliferation of Notch-expressing cells and/or improves survival rate. The present invention has been accomplished on the basis of this finding. Also, the present inventors have found that an anti-nicastrin antibody inhibits reaction between nicastrin and a γ-secretase substrate (i.e., a polypeptide including the intramembrane sequence of a receptor or APP), and thus this reaction system can be employed for selecting, through screening, an antibody which inhibits γ-secretase activity. The present invention has been accomplished also on the basis of this finding.
Accordingly, the present invention provides a therapeutic drug for AD and/or a cancer containing an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative.
The present invention also provides a screening method for selecting an antibody which inhibits γ-secretase activity, characterized by comprising reacting nicastrin with a γ-secretase substrate in a presence of a test antibody.
The present invention also provides use of an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative for producing a therapeutic drug for Alzheimer's disease and/or a cancer.
The present invention also provides a method for treatment of Alzheimer's disease and/or a cancer, characterized by comprising administering an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative to a subject in need thereof.
EFFECTS OF THE INVENTION
According to the therapeutic drug for AD and/or a cancer of the present invention, γ-secretase activity can be inhibited by an anti-nicastrin antibody, to thereby treat Alzheimer's disease and/or a cancer.
According to the screening method of the present invention, the reaction system between nicastrin and a γ-secretase substrate can be employed for selecting an antibody which inhibits γ-secretase activity; i.e., an antibody effective for the treatment of Alzheimer's disease and/or a cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows results of screening of anti-nicastrin antibodies through BV-ELISA.
FIG. 2 shows results of screening of anti-nicastrin antibodies through western blot analysis by use of BV.
FIG. 3 shows results of western blot analysis of various forms of nicastrin expressed in COS-7 cells.
FIG. 4 shows results of an experiment for evaluating cross-reactivity of anti-nicastrin antibodies to deglycosylated nicastrin ("O" represents Endo H-resistant nicastrin; "black dot" represents completely deglycosylated nicastrin; and "Δ" represents neuraminidase-desialylated nicastrin).
FIG. 5 shows results of IP of nicastrin from a soluble membrane fraction of HeLa cell by use of anti-nicastrin antibodies.
FIG. 6 shows results of treatment of a HeLa cell lysate with trypsin.
FIG. 7 shows results of immunostaining of HeLa cells by use of anti-nicastrin antibodies.
FIG. 8 shows results of immunostaining of NKO cells and NKO/NCT cells by use of anti-nicastrin antibodies.
FIG. 9 shows results of co-staining by use of anti-nicastrin antibodies and antibodies to various marker proteins.
FIG. 10 shows results of co-staining by use of anti-nicastrin antibodies and cholera toxin subunit B (CTB).
FIG. 11 shows an effect of anti-nicastrin antibodies on in vitro γ-secretase activity.
FIG. 12 shows an effect of DAPT on a viability of HeLa cells or A549 cells.
FIG. 13 shows an effect of inhibition of nicastrin expression on a viability of A549 cells.
FIG. 14 shows results of inhibition of expression of endogenous nicastrin in A549 cells by siRNA.
FIG. 15 shows an effect of anti-nicastrin antibodies on the viability of A549 cells.
FIG. 16 shows mutation sites of Notch1 gene in various T-ALL-derived cells.
FIG. 17 shows results of western blot analysis of Notch1 gene products in various T-ALL-derived cell lysates.
FIG. 18 shows an effect of a γ-secretase inhibitor YO on proliferation of various T-ALL-derived cells.
FIG. 19 shows an effect of an anti-nicastrin antibody on proliferation of DND-41 cells.
FIG. 20 shows results of western blot analysis of anti-nicastrin antibodies and a nicastrin-N100 fraction.
FIG. 21 shows an effect of anti-nicastrin antibodies on inhibition of binding between nicastrin and N100-FLAG.
FIG. 22A shows an effect of an anti-nicastrin antibody on inhibition of γ-secretase activity in living cells.
FIG. 22B shows an effect of an anti-nicastrin antibody on inhibition of γ-secretase activity in living cells.
FIG. 22C shows an effect of an anti-nicastrin antibody on inhibition of γ-secretase activity in living cells.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will next be described in detail.
The present invention is directed to a therapeutic drug for AD and/or a cancer containing an anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative; to use of the antibody, the derivative, or the fragment for producing such a therapeutic drug; and to a method for the treatment of Alzheimer's disease and/or a cancer.
In the present invention, as described hereinbelow, the anti-nicastrin antibody derivative encompasses a modified anti-nicastrin antibody and an anti-nicastrin antibody to which a compound exhibiting a desired pharmaceutical activity has been bound.
Nicastrin is a membrane protein and forms a complex to exhibit γ-secretase activity. An amino acid sequence of nicastrin and a sequence of the gene coding therefor are disclosed in GenBank number (NM--015331) (SEQ ID Nos: 1 and 2). As used herein, "nicastrin protein" encompasses both full-length nicastrin protein and a fragment of nicastrin. As used herein, "fragment of nicastrin" refers to a polypeptide which includes a predetermined region of nicastrin protein and which does not necessarily have a function of natural nicastrin protein.
Nicastrin protein, which is employed as an antigen in the present invention, is preferably human nicastrin protein, but is not necessarily limited thereto. The nicastrin protein employed in the present invention may be nicastrin derived from any non-human species, such as canine nicastrin, feline nicastrin, mouse nicastrin, hamster nicastrin, or drosophila nicastrin. Preferably, an antibody selected by use of nicastrin protein neutralizes human active γ-secretase including nicastrin as a constituent molecule.
Human active γ-secretase is a large-molecule membrane protein complex having a molecular weight of 250 to 500 kDa or more and including the following four proteins: fragmented presenilin, nicastrin, APH-1, and PEN-2.
The active γ-secretase employed in the present invention may be prepared through any of methods described in the Examples hereinbelow (WO 2005/038023). Natural human active γ-secretase may be prepared from a human brain homogenate, but is very difficult to employ for screening of γ-secretase inhibitors. Therefore, the active γ-secretase employed is preferably prepared through the method by the present inventors for successfully expressing an active γ-secretase complex by use of budding baculovirus (see WO 2005/038023).
In the present invention, γ-secretase activity is determined through a method by Yasuko Takahashi, et al. (J. Biol. Chem. 2003 May 16; 278 (20): 18664-70), which is a generally known method. Specifically, a test substance is mixed with microsomes (serving as an enzyme) prepared from brain tissue or cultured cells, and the mixture is incubated at 4° C. for 12 hours. Subsequently, 1 μM C100FmH serving as a substrate is added to the reaction mixture, and the mixture is incubated at 37° C. for 12 hours. Thereafter, an amount of amyloid-β is measured through sandwich ELISA, to thereby determine γ-secretase activity.
Preparation of Anti-Nicastrin Antibody
Preferably, the anti-nicastrin antibody employed in the present invention not only binds specifically to nicastrin protein, but also neutralizes human active γ-secretase. No particular limitation is imposed on an origin, type (monoclonal or polyclonal), and form of the anti-nicastrin antibody. Specifically, the anti-nicastrin antibody may be a known antibody such as a mouse antibody, a rat antibody, an avian antibody, a human antibody, a chimera antibody, and a humanized (CDR-grafted) antibody. The anti-nicastrin antibody is preferably a human, chimera, or humanized monoclonal antibody.
Examples of the anti-nicastrin monoclonal antibody include a monoclonal antibody produced by a hybridoma, and a monoclonal antibody produced in a host transformed with an expression vector containing a gene for the antibody through a genetic engineering technique.
Basically, a hybridoma which produces the monoclonal antibody may be prepared through a known technique as described below. Specifically, the hybridoma may be prepared through the following procedure: a mammal is immunized with nicastrin protein serving as a sensitizing antigen through a customary immunization method; the resultant immunocyte is fused with a known parental cell through a customary cell fusion method; and a cell for producing the monoclonal antibody is selected through a customary screening method.
Specifically, the monoclonal antibody can be prepared as follows.
Firstly, nicastrin protein, which is employed as a sensitizing antigen for preparing the monoclonal antibody, is obtained through expression of a nicastrin gene/amino acid sequence disclosed in GenBank number (NM--015331). Specifically, an appropriate host cell is transformed with a known expression vector system containing the gene sequence encoding nicastrin, and then human nicastrin protein of interest is purified from the resultant host cell or a culture supernatant of the cell through a known method. Alternatively, natural nicastrin protein may be employed after being purified.
Subsequently, the thus-purified nicastrin protein is employed as a sensitizing antigen. Alternatively, a partial peptide of the nicastrin protein may be employed as a sensitizing antigen. Such a partial peptide may be obtained through chemical synthesis on the basis of the amino acid sequence of nicastrin protein, through integration of a portion of the nicastrin gene into an expression vector, or through degradation of natural nicastrin protein by use of protease. No particular limitation is imposed on a site or size of a nicastrin protein portion employed as a partial peptide.
No particular limitation is imposed on the mammal which is immunized with the sensitizing antigen, but preferably, the mammal is selected in consideration of compatibility of the resultant immunocyte with a parental cell employed for cell fusion. In general, a rodent (e.g., mouse, rat, or hamster), avian, rabbit, monkey, or the like is employed.
Immunization of an animal with the sensitizing antigen is carried out through a known method. For example, in a generally employed immunization method, the sensitizing antigen is intraperitoneally or subcutaneously injected into a mammal. Specifically, the sensitizing antigen is diluted by PBS (phosphate-buffered saline), saline, or the like, to form a suspension of an appropriate volume. If desired, the resultant suspension is mixed with an appropriate volume of a common adjuvant (e.g., Freund's complete adjuvant). After emulsification of the resultant mixture, the emulsion is administered to a mammal several times every 4 to 21 days. Upon immunization with the sensitizing antigen, an appropriate carrier may be employed. Particularly when the sensitizing antigen is a partial peptide of low molecular weight, preferably, the partial peptide employed for immunization is bound to a carrier protein such as albumin or keyhole limpet hemocyanin.
After immunization of a mammal as described above, and following confirmation of an increase in serum level of an antibody of interest, immunocytes are collected from the mammal and then subjected to cell fusion. The type of immunocytes is particularly preferably splenocyte.
A mammalian myeloma cell is employed as a parental cell which is fused with the aforementioned immunocyte. The myeloma cell employed is preferably a known cell line; for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D. H., et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M., et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F., et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), or R210 (Galfre, G., et al., Nature (1979) 277, 131-133).
Cell fusion between the aforementioned immunocyte and myeloma cell may be basically carried out through a known method, such as a method of Kohler, Milstein, et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).
More specifically, the aforementioned cell fusion is carried out in a common nutrient culture medium in the presence of, for example, a cell fusion promoter. Examples of the cell fusion promoter employed include polyethylene glycol (PEG) and Sendai virus (HVJ). If desired, an auxiliary agent (e.g., dimethyl sulfoxide) may be further added in order to enhance cell fusion efficiency.
The ratio of the immunocyte and myeloma cell employed may be determined as desired. For example, an amount of the immunocyte is preferably 1 to 10 times that of the myeloma cell. Examples of the culture medium which may be employed for the aforementioned cell fusion include RPMI 1640 medium and MEM medium, which are suitable for proliferation of the aforementioned myeloma cell line; and culture media which are generally employed for such a cell culture. Such a culture medium may be employed in combination with a serum supplement such as fetal calf serum (FCS).
In the cell fusion, predetermined amounts of the aforementioned immunocyte and myeloma cell are well-mixed in any of the aforementioned culture media, and a solution of PEG (e.g., PEG having an average molecular weight of about 1,000 to about 6,000) which has been heated in advance to about 37° C. is added to the resultant mixture in a predetermined amount (generally 30 to 60% (w/v)), followed by mixing, to thereby yield a hybridoma of interest. Subsequently, a procedure including sequential addition of an appropriate culture medium and removal of a supernatant obtained through centrifugation is repeated, to thereby remove substances (e.g., a cell fusion promoter) which are not suitable for growth of the hybridoma.
Separation of the thus-yielded hybridoma is carried out through culturing in a common selective culture medium such as a HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). A culturing in the aforementioned HAT medium is continued for a sufficient period of time (generally several days to several weeks) for apoptosis of cells (i.e., non-fused cells) other than the hybridoma of interest. Subsequently, a customary limiting dilution technique is performed for screening and monocloning of the hybridoma which produces a target antibody.
An antibody which recognizes nicastrin protein may be prepared through a method described in WO 03/104453.
Screening and monocloning of a target antibody may be carried out through a known screening method on a basis of antigen-antibody reaction. For example, an antigen is bound to a carrier (e.g., beads made of polystyrene or a similar material, or a commercially available 96-well microtiter plate) and then reacted with a culture supernatant of the hybridoma, and subsequently the carrier is washed, followed by reaction with, for example, an enzyme-labeled secondary antibody, to thereby determine whether or not the culture supernatant contains a target antibody which reacts with a sensitizing antigen. Cloning of the hybridoma which produces a target antibody may be performed through, for example, a limiting dilution technique. In this case, the antigen may be an antigen employed in immunization.
In addition to preparation of the aforementioned hybridoma through immunization of a non-human animal with an antigen, a human antibody of interest having binding activity to nicastrin may be prepared by sensitizing human lymphocyte with nicastrin in vitro, and fusing the thus-sensitized lymphocyte with a human-derived myeloma cell having permanent division capacity (see JP-A-01-059878). Alternatively, nicastrin serving as an antigen may be administered to a transgenic animal having all of the human antibody gene repertories, to thereby yield a cell which produces an anti-nicastrin antibody, and a human antibody against nicastrin may be obtained from the cell after it has been immortalized (see WO 94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).
The thus-prepared monoclonal-antibody-producing hybridoma can be subcultured in a common culture medium and can be stored in liquid nitrogen for a long period of time.
A monoclonal antibody is produced from the hybridoma through, for example, a method in which the hybridoma is cultured by a customary technique, and the monoclonal antibody is obtained from the resultant culture supernatant; or a method in which the hybridoma is administered to a mammal exhibiting compatibility with the hybridoma to thereby proliferate the hybridoma, and the monoclonal antibody is obtained from ascitic fluid of the mammal. The former method is suitable for obtaining a monoclonal antibody of high purity, whereas the latter method is suitable for a mass production of a monoclonal antibody.
The monoclonal antibody employed in the present invention may be a recombinant antibody. Such a recombinant antibody is produced through the following procedure: the antibody gene is cloned from the hybridoma; the gene is integrated into an appropriate vector; and the vector is introduced into a host, followed by production of the recombinant antibody through a genetic recombination technique (see, for example, Vandamme, A. M., et al., Eur. J. Biochem. (1990) 192, 767-775, 1990).
Specifically, mRNA encoding a variable (V) region of an anti-nicastrin antibody is isolated from the hybridoma which produces the anti-nicastrin antibody. Isolation of mRNA is carried out as follows. Total RNA is prepared through a known method such as the guanidine ultracentrifugation method (Chirgwin, J. M., et al., Biochemistry (1979) 18, 5294-5299) or the AGPC method (Chomczynski, P., et al., Anal. Biochem. (1987) 162, 156-159), and target mRNA is prepared by means of, for example, mRNA Purification Kit (product of Pharmacia). Alternatively, mRNA may be directly prepared by means of QuickPrep mRNA Purification Kit (product of Pharmacia).
The thus-obtained mRNA is employed for synthesis of cDNA of the antibody V region by use of reverse transcriptase. Synthesis of cDNA is carried out by means of, for example, AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (product of Seikagaku Corporation). Alternatively, synthesis and amplification of cDNA may be carried out by means of, for example, 5'-Ampli FINDER RACE Kit (product of Clontech) or the 5'-RACE method using PCR (Frohman, M. A., et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A., et al., Nucleic Acids Res. (1989) 17, 2919-2932).
A target DNA fragment is purified from the resultant PCR product and ligated to vector DNA. Subsequently, a recombinant vector is prepared from the vector DNA and then introduced into Escherichia coli or the like, followed by colony selection, to thereby prepare a recombinant vector of interest. The nucleotide sequence of the target DNA fragment is determined through a known method such as a dideoxynucleotide chain termination method.
DNA encoding the V regions of a target anti-nicastrin antibody is obtained, and then the DNA is integrated into an expression vector containing DNA encoding constant regions (C regions) of the target antibody.
In order to produce the anti-nicastrin antibody employed in the present invention, the gene for the antibody is integrated into an expression vector so that the gene can be expressed under a control of an expression regulatory region (e.g., an enhancer or a promoter). Subsequently, a host cell is transformed with this expression vector for expression of the antibody.
The gene for the antibody may be expressed by transforming a host cell with both an expression vector containing the DNA encoding a heavy chain (H chain) of the antibody and an expression vector containing the DNA encoding a light chain (L chain) of the antibody, or by transforming a host cell with a single expression vector containing the DNA encoding the heavy and light chains of the antibody (see WO 94/11523).
In addition to the aforementioned host cell, a transgenic animal may be employed for production of a recombinant antibody. For example, an antibody gene is inserted into a gene encoding a protein produced specifically in milk (such as goat β-casein) to prepare a fusion gene. A DNA fragment including the fusion gene having the inserted antibody gene is injected into an embryo of a goat, and this embryo is implanted into a female goat. An antibody of interest is obtained from milk produced by transgenic goats born from the goat impregnated with the embryo or progeny thereof. In order to increase an amount of the antibody-containing milk produced by the transgenic goats, hormones may be administered to the transgenic goats as appropriate (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
In the present invention, in addition to the aforementioned antibodies, an artificially modified, genetically recombinant antibody (e.g., a chimera antibody or a humanized antibody) may be employed. Such a modified antibody may be produced through a known method.
Specifically, a chimera antibody is prepared through the following procedure: the above-obtained DNA encoding the antibody V regions is ligated to the DNA encoding the human antibody C regions; the thus-ligated DNA is integrated into an expression vector; and the expression vector is introduced into a host for production of the chimera antibody. Through this known procedure, a chimera antibody useful for the present invention can be prepared.
A humanized antibody is also called a "reshaped human antibody" and is obtained by grafting a complementarity-determining regions (CDRs) of an antibody from a non-human mammal (e.g., mouse) into the complementarity-determining regions of a human antibody. Typical gene recombination techniques for preparing such a humanized antibody are known (see European Patent Application Laid-Open (EP) No. 125023 and WO 96/02576).
Specifically, a DNA sequence designed to ligate a CDRs of a mouse antibody to a framework regions (FRs) of a human antibody is synthesized through PCR employing, as primers, several oligonucleotides prepared to have portions overlapping terminal regions of both the CDRs and FRs (see the method described in WO 98/13388).
The framework regions of the human antibody ligated via the CDRs are selected in such a manner that the complementarity-determining regions form a proper antigen-binding site. If necessary, amino acid residues in the framework regions of the antibody variable regions may be substituted so that the complementarity-determining regions of a reshaped human antibody form a proper antigen-binding site (Sato, K., et al., Cancer Res. (1993) 53, 851-856).
The C regions employed in a chimera antibody or a humanized antibody may be those of a human antibody; for example, Cγ1, Cγ2, Cγ3, and Cγ4 in the H chain, and Cκ and Cλ in the L chain. The C regions of the human antibody may be modified so as to improve a stability of the antibody or to achieve stable production thereof.
The chimera antibody includes the variable regions of an antibody derived from a non-human mammal and the constant regions of a human antibody. Meanwhile, the humanized antibody includes the complementarity-determining regions of an antibody derived from a non-human mammal and the framework regions and C regions of a human antibody. The humanized antibody is useful as an active ingredient of a therapeutic agent, since it exhibits low antigenicity in a human body.
The anti-nicastrin antibody employed in the present invention is not limited to the whole antibody molecule. So long as the anti-nicastrin antibody binds to nicastrin protein, the antibody may be an antibody fragment, derivatives of the antibody (including a modified antibody, and an antibody bound to a compound exhibiting a desired pharmaceutical activity), a divalent antibody, or a monovalent antibody. The anti-nicastrin antibody is preferably an antibody which neutralizes human active γ-secretase.
Examples of the antibody fragment include Fab, F(ab')2, Fab/c having Fab having one Fv and complete Fc, and single-chain Fv (scFv) in which Fv fragments of the H or L chain are linked together with an appropriate linker. Specifically, an antibody is treated with an enzyme (e.g., papain or pepsin) to produce an antibody fragment. Alternatively, a gene encoding such an antibody fragment is constructed and introduced into an expression vector, followed by expression in an appropriate host cell (see, for example, Co, M. S., et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J., et al., Methods in Enzymology (1989) 121, 663-669; and Bird, R. E., et al., TIBTECH (1991) 9, 132-137).
A single-chain Fv (scFv) is obtained by linking the H chain V region and L chain V region of an antibody. In the scFv fragment, the H chain V region and the L chain V region are linked by a linker (preferably, a peptide linker) (Huston, J. S., et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H chain V region and the L chain V region in the scFv fragment may be derived from any of the antibodies described herein. The peptide linker employed for linking the V regions is, for example, any single-stranded peptide including 12 to 19 amino acid residues.
DNA encoding the scFv fragment is obtained through PCR amplification employing, as a template, an entire sequence of the DNA encoding the H chain or H chain V region of the aforementioned antibody or the DNA encoding the L chain or L chain V region of the antibody, or a portion of the DNA sequence encoding an amino acid sequence of interest, in combination with a primer pair defining both ends of the DNA sequence, followed by amplification employing the DNA encoding a peptide linker region in combination with a primer pair which defines both ends of the DNA so that the respective ends are linked to the H and L chains.
Once the DNA encoding the scFv fragment is prepared, an expression vector containing the DNA and a host transformed with the expression vector can be obtained through a customary method, and the scFv fragment can be obtained through a customary method by use of the host.
Such an antibody fragment may be produced by a host after a gene for the fragment has been obtained and expressed in a manner similar to that described above. As used herein, the term "antibody" also encompasses such an antibody fragment.
Also, a modified anti-nicastrin antibody prepared through conjugation of a molecule (e.g., polyethylene glycol (PEG) or a sugar chain) to an anti-nicastrin antibody may be employed. Through such modification, a half-life of the anti-nicastrin antibody can be prolonged, and hydrolysis or elimination thereof can be reduced in blood. As used herein, the term "antibody" also encompasses such a modified antibody. Such a modified antibody may be prepared through chemical modification of the above-obtained antibody or a fragment thereof. Methods for modifying antibodies have already been established in the art.
Also, the antibody employed in the present invention may be a bispecific antibody. The bispecific antibody may have antigen-binding sites recognizing different epitopes of NCT molecule. A bispecific antibody may be prepared by binding HL pairs of two antibodies, or may be obtained from a bispecific-antibody-producing fused cell prepared through fusion of hybridomas producing different monoclonal antibodies. Alternatively, a bispecific antibody may be prepared through a genetic engineering technique.
The above-constructed gene for the antibody may be expressed through a known method, to thereby yield the antibody. In the case where a mammalian cell is employed, the antibody gene may be expressed by functionally binding a common useful promoter, the gene which is expressed, and a polyA signal downstream of a 3'-end thereof. Examples of the promoter/enhancer which may be employed include human cytomegalovirus immediate early promoter/enhancer.
Other promoters/enhancers which may be employed for antibody expression in the present invention include viral promoters/enhancers such as retrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40); and promoters/enhancers derived from mammalian cells, such as human elongation factor 1α (HEF1α).
When SV40 promoter/enhancer is employed, gene expression can be readily carried out through a method of Mulligan, et al. (Nature (1979) 277, 108), whereas when HEF1α promoter/enhancer is employed, gene expression can be readily carried out through a method of Mizushima, et al. (Nucleic Acids Res. (1990) 18, 5322).
In the case where Escherichia coli are employed, the gene for the antibody can be expressed by functionally binding a common useful promoter, a signal sequence for secreting the antibody, and the antibody gene which is expressed. Examples of the promoter which may be employed include lacZ promoter and araB promoter. When lacZ promoter is employed, the gene can be expressed through a method of Ward, et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), whereas when araB promoter is employed, the gene can be expressed through a method of Better, et al. (Science (1988) 240, 1041-1043).
When the antibody is produced in a periplasm of Escherichia coli, a pe1B signal sequence (Lei, S. P., et al., J. Bacteriol. (1987) 169, 4379) may be employed as a signal sequence for secreting the antibody. The antibody produced in the periplasm is isolated and then employed by appropriately refolding a structure of the antibody.
Replication origins which may be employed include those derived from SV40, polyomavirus, adenovirus, bovine papilloma virus (BPV). In order to increase gene copy number in a host cell system, the expression vector employed may contain a selective marker such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.
Any expression system such as a eukaryotic or prokaryotic system may be used for production of the antibody employed in the present invention. Examples of the eukaryotic cell include animal cells of, for example, established mammalian cell line, cells of insect cell line, filamentous fungal cells, and yeast cells; and examples of the prokaryotic cell include cells of a bacterium such as Escherichia coli.
Preferably, the antibody employed in the present invention is expressed in a mammalian cell such as CHO, COS, myeloma, BHK, Vero, or HeLa cell.
Subsequently, the above-transformed host cell is cultured in vitro or in vivo to produce a target antibody. Culturing of the host cell is carried out through a known method. For example, DMEM, MEM, RPMI 1640, or IMDM may be employed as a culture medium, and a serum supplement such as fetal calf serum (FCS) may be employed in combination.
The above-expressed or produced antibody can be isolated from cells or a host animal and purified to homogeneity. Isolation and purification of the antibody employed in the present invention may be carried out by means of an affinity column. Examples of columns employing protein A column include Hyper D, POROS, and Sepharose F.F. (products of Pharmacia). No particular limitation is imposed on the method for isolation/purification of the antibody, and the antibody may be isolated or purified through any method which is generally employed for isolation/separation of proteins. For example, the antibody may be isolated/purified by appropriately selecting or combining chromatography columns other than the aforementioned affinity columns, filters, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
As described in the Examples hereinbelow, the above-obtained anti-nicastrin antibody recognizes nicastrin protein (i.e., a constituent molecule of human active γ-secretase), binds specifically to nicastrin protein, and exhibits an activity to neutralize γ-secretase. In addition, the anti-nicastrin antibody has an ability to inhibit proliferation of γ-secretase-dependent cancer cells. Therefore, the anti-nicastrin antibody, a derivative of the antibody, or a fragment of the antibody or the derivative is effective as a therapeutic drug for Alzheimer's disease and/or a cancer.
Conceivably, the cancer which can be treated by the present invention is a nicastrin-expressing cancer and/or a γ-secretase-dependent cancer.
Examples of such a cancer include lung cancer and T-cell acute lymphoblastic leukemia.
As used herein, "nicastrin-expressing cancer" refers to a cancer in which nicastrin protein is produced through expression of the nicastrin gene; and "γ-secretase-dependent cancer" refers to a cancer in which proliferation of cancer cells requires γ-secretase, and cancer cell proliferation is inhibited or cancer cells die through inhibition of γ-secretase activity.
The anti-nicastrin antibody derivative or a fragment thereof also encompasses a product prepared by conjugating a compound exhibiting a desired pharmaceutical activity to the anti-nicastrin antibody or a fragment thereof through a customary method. Such an anti-nicastrin antibody derivative may be employed in, for example, a missile therapy specifically targeting nicastrin. As used herein, "compound exhibiting a desired pharmaceutical activity" refers to a compound exhibiting, for example, a pharmaceutical activity to inhibit or promote a substance (e.g., an enzyme or a receptor) which directly or indirectly causes symptoms to progress.
Examples of compounds exhibiting a desired pharmaceutical activity for cancer treatment include a compound which causes damage to cancer cells, and a compound which provides or enhances cytotoxic activity (e.g., a radioisotope). The radioisotope employed may be any radioisotope known to those skilled in the art, but is preferably 131I, 99mTc, 111In, or 90Y.
Cancer treatment employing an antibody bound to a radioisotope-containing compound may be carried out through a method known to those skilled in the art. Specifically, firstly, a small amount of an antibody bound to a radioisotope-containing compound is administered to a patient, followed by whole-body scintigraphy. After determination that a degree of binding between the antibody and normal tissue cells is low but the degree of binding between the antibody and cancer cells is high, a large amount of the radioisotope-bound antibody is administered to the patient.
The therapeutic drug of the present invention may be prepared into a drug product by subjecting both the drug and a pharmaceutically acceptable carrier well known in the art to a drug preparation process such as mixing, dissolution, granulation, tableting, emulsification, encapsulation, or lyophilization.
For oral administration, the therapeutic drug of the present invention may be mixed with, for example, a pharmaceutically acceptable solvent, excipient, binder, stabilizer, or dispersant, and the mixture may be prepared into a dosage form such as tablet, pill, sugar-coated agent, soft capsule, hard capsule, solution, suspension, emulsion, gel, syrup, or slurry.
For parenteral administration, the therapeutic drug of the present invention may be mixed with, for example, a pharmaceutically acceptable solvent, excipient, binder, stabilizer, or dispersant, and the mixture may be prepared into a dosage form such as injection solution, suspension, emulsion, cream, ointment, inhalant, or suppository. For formulation of an injection, the therapeutic drug of the present invention may be dissolved in an aqueous solution, preferably, a physiologically compatible buffer (e.g., Hanks' solution, Ringer solution, or saline buffer). The composition may be in the form of suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the therapeutic drug may be produced in the form of powder, and, before use, the drug may be prepared into an aqueous solution or suspension with, for example, sterile water. For inhalation administration, the therapeutic drug of the present invention may be powdered and may be prepared into a powder mixture together with an appropriate base such as lactose or starch. For production of a suppository, the therapeutic drug of the present invention may be mixed with a conventional suppository base such as cocoa butter. The therapeutic drug of the present invention may be formulated into a sustained-release drug product by encapsulating the drug in, for example, a polymer matrix.
A dose of the therapeutic drug of the present invention or a number of doses thereof varies depending on a dosage form or administration route thereof, or the symptom, age, or body weight of a patient in need thereof. The therapeutic drug can be administered once to several times per day so that a daily dose of the drug is generally about 0.001 mg to about 1,000 mg per kg body weight, preferably about 0.01 mg to about 10 mg per kg body weight.
Generally, the therapeutic drug is administered through a parenteral route; for example, injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection), or transdermal, transmucosal, transnasal, or transpulmonary administration. However, no particular limitation is imposed on the administration route of the therapeutic drug, and the drug may be orally administered.
A screening method for selecting an antibody which inhibits γ-secretase activity.
As described in the Examples hereinbelow, an anti-nicastrin antibody has been found to inhibit reaction between nicastrin and a γ-secretase substrate (e.g., C99 or N99).
Therefore, the screening method of the present invention for selecting an antibody which inhibits γ-secretase activity is a promising method for searching a therapeutic drug for AD or cancer.
In the screening method of the present invention, nicastrin is reacted with a γ-secretase substrate (e.g., a polypeptide formed of the entirety or a portion of Notch receptor and/or APP (including the intramembrane sequence)) in the presence of a test antibody, and the reaction between nicastrin and the substrate is detected.
Specifically, nicastrin is reacted with a polypeptide formed of the entirety or a portion of the sequence of Notch or APP in the presence of a test antibody through addition thereof, and whether or not the added test antibody inhibits the reaction is determined through a known detection method.
Alternatively, a test antibody is exposed to cells expressing nicastrin and a polypeptide formed of the entirety or a portion of the sequence of Notch and/or APP, and whether or not a product is produced through the reaction between nicastrin and the polypeptide is determined through a known detection method.
Preferably, the latter screening method is carried out. The latter screening method requires a simpler screening process. In addition, when the latter screening method is carried out in combination with a known detection method, numerous test antibodies can be screened to determine whether or not they inhibit γ-secretase activity within a short period of time. Thus, a therapeutic drug for AD or cancer can be developed within a short period.
The γ-secretase substrate employed in the screening method may be a polypeptide formed of the entirety or a portion of Notch receptor (NM--008714) (SEQ ID NO: 3) (including the intramembrane sequence) and/or a polypeptide formed of the entirety or a portion of APP protein (NM--000484) (SEQ ID NO: 4) (including the intramembrane sequence). The polypeptide formed of the entirety or a portion of Notch receptor or APP protein may be prepared through expression of a gene having a sequence (5'-cacctcatgtacgtggcagcggccgccttcgtgctcctgttctttgtgggctgtggggtg- c tgctg-3') (SEQ ID NO: 6) and encoding a polypeptide including the intramembrane sequence of Notch receptor (NH2-HLMYVAAAAFVLLFFVGCGVLL-COOH) (SEQ ID NO: 5), or a gene having a sequence (5'-ggtgcaatcattggactcatggtgggcggtgttgtcatagcgacagtgatcgtcatcacc- t tggtgatgctg-3') (SEQ ID NO: 8) and encoding a polypeptide including the intramembrane sequence of APP protein (NH2-GAIIGLMVGGVVIATVIVITLVML-COOH) (SEQ ID NO: 7). Particularly preferably, the polypeptide formed of the entirety or a portion of Notch receptor or APP protein is prepared through expression of a gene having a sequence (SEQ ID NO: 10) and encoding 99 amino acid residues (No. 1711 to No. 1809) of a protein of Notch receptor including the intramembrane sequence (NH2-VKSEPVEPPLPSQLHLVYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFK- VSEASKK KRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLE-COOH) (SEQ ID NO: 9) (the 99 amino acid residues may be called "N99"), or a gene having a sequence (SEQ ID NO: 12) and encoding 99 amino acid residues (the C-terminus to No. 99) of a protein of APP including the intramembrane sequence (NH2-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIH HGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN-COOH) (SEQ ID NO: 11) (the 99 amino acid residues may be called "IC99").
The polypeptide serving as a γ-secretase substrate, which is formed of the entirety or a portion of a protein, is obtained from the corresponding amino acid sequence or expressing a gene encoding the amino acid residues. Alternatively, the polypeptide is obtained from a natural product.
The polypeptide serving as a γ-secretase substrate, which is formed of the entirety or a portion of a protein, is preferably derived from human. However, the origin of the polypeptide is not limited to human, and the polypeptide may be derived from any non-human species such as dog, cat, mouse, hamster, or drosophila.
The amino acid sequence of nicastrin or a γ-secretase substrate or the sequence of the gene coding therefor may be provided, before expression thereof, with a tag sequence (e.g., V5 or FLAG sequence), which is selected in consideration of a detection method employed.
Whether or not a test antibody inhibits γ-secretase activity may be determined through a known technique such as co-immunoprecipitation (IP), western blotting, ELISA, reporter gene assay, a SPA beads method, a fluorescence polarization method, or a homogeneous time-resolved fluorescence method. These techniques may be employed singly or in combination as appropriate.
For example, co-immunoprecipitation (IP) and western blotting may be employed in combination. In this case, the amino acid sequence of nicastrin or a peptide formed of the entirety or a portion of Notch receptor or APP (including the intramembrane sequence) or the sequence of the gene coding therefor is provided with a tag sequence (e.g., FLAG or V5 sequence) through a known method, and the protein or peptide is expressed in a host cell.
Nicastrin or the peptide formed of the entirety or a portion of Notch receptor or APP (including the intramembrane sequence) is extracted from the host cell by a known extraction method including lysis of the cell membrane, followed by purification as appropriate.
The thus-extracted nicastrin is diluted with a culture medium and then mixed with a test antibody, and reaction is carried out at 4° C. for 8 to 12 hours. Thereafter, the Notch or APP peptide is added to the reaction mixture, followed by further mixing for three to four hours. A HEPES buffer containing 0.5% CHAPSO is employed as a buffer solution.
An antibody corresponding to the tag is added, and IP is carried out. Subsequently, a precipitated fraction is analyzed through a known western blot technique. The tag-corresponding antibody may be bound to a carrier (e.g., agarose beads) in advance.
In this case, when nicastrin and the peptide formed of the entirety or a portion of Notch receptor or APP (including the intramembrane sequence) are precipitated in smaller amounts, the test antibody is determined to have higher percent inhibition of γ-secretase activity.
Alternatively, binding assay may be carried out by immobilizing one of nicastrin and the peptide on, for example, a carrier or an assay plate, and labeling the other with, for example, a radioisotope or a fluorescent substance. A test antibody detected may be provided with a tag (e.g., an antigen) or a label (e.g., a radioisotope).
Whether or not a test antibody inhibits γ-secretase activity may be determined through, for example, a method employing a GAL4-UAS system and ELISA or a reporter gene in combination. In this case, a construct (SC100G) is prepared by inserting GAL4 into C99 through a known method, and a reporter construct (UAS-luc) is prepared by inserting a UAS sequence into an upstream region of the luciferase gene serving as a reporter gene. These constructs are introduced into host cells through a known technique such as lipofection. Cells constitutively expressing nicastrin are selected by use of, for example, an antibiotic-resistant marker as appropriate.
The constitutively expressing cells are cultured at 37° C. for 24 hours, and then a test antibody is exposed to the cells, followed by expression of the transgene. Expression of the gene is induced by addition of 10 mM n-butylic acid. After culturing at 37° C. for 12 hours, the cells or the resultant supernatant is recovered.
In the case where the cells are employed, when the amount of luminescence generated by luciferase is reduced after lysis of the cells, the test antibody is determined to inhibit binding between nicastrin and a Notch receptor intramembrane peptide or an APP peptide, and to inhibit cleavage of the Notch receptor intramembrane peptide or the APP peptide; i.e., the test antibody is determined to have high percent inhibition of γ-secretase activity.
In the case where the supernatant is employed, an extracellularly released Aβ peptide fraction having an indicator applicable to ELISA is assayed through ELISA. When the degree of ELISA reaction is low, the test antibody is determined to inhibit binding between nicastrin and Notch receptor or an APP peptide, and to inhibit cleavage of the intramembrane sequence of a Notch receptor peptide or the APP peptide; i.e., the test antibody is determined to have high percent inhibition of γ-secretase activity.
Alkaline phosphatase or GFP may be employed in place of luciferase.
The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.
Culturing of Insect Cells
Insect cells (Spodoptera frugiperda, Sf9) were cultured at 27° C. by use of Grace's Insect Media Supplemented (Invitrogen) containing 10% fetal bovine serum (FBS, Sigma), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen). When mass culture was carried out, 0.001% pluronic F-68 (Invitrogen) was added to the aforementioned medium placed in a 1-L spinner flask.
Preparation of Recombinant Virus
Human nicastrin cDNA cloned into pEF6-TOPO/V5-His (Invitrogen) (pEF6-NCT) (T. Tomita et al., FEBS Lett. 520 (2002) 117-121) was subcloned into pBlueBac4.5 (Invitrogen) so that the V5-His tag derived from the vector was provided on the C-terminal side, to thereby prepare a human-nicastrin-containing construct (pBlueBac4.5-NCT). Recombinant virus preparation was carried out according to a protocol attached Bac-N-Blue Transfection Kit (Invitrogen). Specifically, Sf9 cells were transfected with Bac-N-Blue DNA and the above-prepared plasmid (4 μg), followed by purification through a plaque assay (several times), to thereby prepare a recombinant virus containing only a target gene. After preparation of a high titer stock, a titer of the virus was determined through a plaque assay.
Confirmation of Expression of Nicastrin on BV
Expression of nicastrin (i.e., a single-transmembrane protein) on BV was confirmed by use of the above-prepared recombinant virus. Sf9 cells were infected with the recombinant virus at a multiplicity of infection (MOI) of 5, and the cells and BV were recovered after 12 hours, 24 hours, 48 hours, or 72 hours initiation of infection, followed by confirmation of expression of nicastrin through immunoblotting by use of an anti-nicastrin N-terminal antibody (anti-NCT (N-19), SantaCruz) and an anti-His antibody. As a result, nicastrin was found to be sufficiently expressed in both a cell fraction and a BV fraction 48 hours after initiation of infection. This indicates that, similar to the case of SREBP-2 (Y. Urano, et al., Biochem. Biophys. Res. Commun. 308 (2003) 191-196), nicastrin is expressed on BV.
Preparation of Anti-Nicastrin Antibody by Use of Budding Virus (BV)
Since a large amount of gp64, which is a virus-derived membrane protein and exhibits high antigenicity, is expressed on BV, when a mouse is infected with BV, an anti-gp64 antibody is strongly induced, and difficulty is encountered in yielding an antibody to a target antigen. Therefore, gp64 transgenic mice, which were prepared so as to exhibit resistance to gp64, were employed as mice for immunization.
Sf9 cells (5×108 cells/500 mL) were infected with human-nicastrin-expressing recombinant virus (NCT-BV) at an MOI of 5, and cultured for 48 hours, followed by recovery of a culture supernatant. BV serving as an antigen was prepared from the culture supernatant through ultracentrifugation, and then gp64 transgenic mice were immunized five times with the antigen.
Screening of antisera and a resultant hybridoma culture supernatants was carried out through BV-ELISA by a customary method. There were added NCT-BV employed during immunization (serving as an antigen for immobilization), and SREBP+SCAP-BV prepared through coinfection of SREBP-2 and SREBP cleavage-activating protein (SCAP) (Y. Urano, et al., Biochem. Biophys. Res. Commun. 308 (2003) 191-196) or wild-BV containing no foreign gene (20 μg/mL in saline) (serving as a negative control) (50 μL/well). As a result, there were yielded many clones which do not respond to wild-type BV or SREBP/SCAP-expressing BV but show positive response to only nicastrin-expressing BV (FIG. 1). Similarly, there were yielded a plurality of clones (e.g., PPMX0401 and PPMX0410) which recognize nicastrin expressed on BV, as determined through immunoblotting employing BV (FIG. 2).
COS-7 cells (cells derived from simian kidney), HeLa cells (cells derived from human cervical cancer), A549 cells (cells derived from human lung cancer), or NKO cells (fibroblasts derived from nicastrin knockout mouse: T. Li, et al., J. Neurosci. 23 (2003) 3272-3277) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen) at 37° C. and 5% CO2.
Identification of Antibody by Use of BV and Forced Expression Product
The culture supernatants of positive clones selected through BV-ELISA were subjected to SDS-PAGE and immunoblotting by use of NCT-BV, Wild-BV, human wild-type nicastrin, and mutant forms of nicastrin in the presence of a 1×SDS-PAGE sample buffer. An anti-nicastrin N-terminal antibody (N-19) was employed as a positive control. In transient expression by use of animal cells, transfection into COS-7 cells was carried out by use of DEAE-dextran, and cells were recovered 48 hours after initiation of transfection. pEF6-NCT was employed for human wild-type nicastrin. Mutant nicastrin constructs (Δ312 and Δ694) were prepared from pEF6-NCT through long PCR (T. Tomita, et al., FEBS Lett. 520 (2002) 117-121).
As a result, all the tested antibodies were found to recognize exogenously expressed human wild-type nicastrin. The anti-nicastrin N-terminal antibody (N-19) (i.e., a positive control) or an antibody to the C-terminal-added V5 tag recognized both wild-type nicastrin and mutant forms of nicastrin. In contrast, almost all the above-prepared antibodies (clones) (e.g., PPMX0401 and PPMX0410) did not recognize nicastrin Δ312 (FIG. 3). This suggests that the epitope site of each of the above-prepared antibodies is present in the extracellular domain of nicastrin.
Preparation of Cells Constitutively Expressing Nicastrin
For the purpose of analysis of anti-nicastrin antibodies, NKO cells were transfected with pEF6-NCT by use of LipofectAmine (Invitrogen), and then NKO cells constitutively expressing human nicastrin (NKO/NCT cells) were selected in a medium containing 10 μg/mL blasticidin.
Deglycosylation of Nicastrin
As has been known, nicastrin has, in the sequence thereof, 20 potential glycosylation sites and highly undergoes N-linked glycosylation (T. Tomita, et al., FEBS Lett. 520 (2002) 117-121; J. Y. Leem, et al., J. Biol. Chem. 277 (2002) 19236-19249; D. S. Yang, et al., J. Biol. Chem. 277 (2002) 28135-28142; and W. T. Kimberly, et al., J. Biol. Chem. 277 (2002) 35113-35117).
Nicastrin is classified, on the basis of the degree of glycosylation, into mature nicastrin (molecular weight: about 130 kDa) and immature nicastrin (molecular weight: about 110 kDa). Active γ-secretase complex contains only mature nicastrin. Among N-linked sugar chains, complex-type sugar chains are known to exhibit resistance to endoglycosidase H (Endo H) but to be cleaved by peptide: N-glycosidase F (PNGase). Therefore, through Endo H treatment, the molecular weight of complex-type glycosylated mature nicastrin is reduced to about 115 kDa, whereas the molecular weight of immature nicastrin having no complex-type sugar chain is reduced to about 80 kDa. In contrast, through PNGase F treatment, the molecular weights of both the mature nicastrin and immature nicastrin are reduced to about 80 kDa (D. S. Yang, et al., J. Biol. Chem. 277 (2002) 28135-28142, and W. T. Kimberly, et al., J. Biol. Chem. 277 (2002) 35113-35117).
In Example 8, a deglycosylation experiment was carried out for a purpose of epitopic analysis of anti-nicastrin antibodies.
Firstly, NKO/NCT cells were washed with PBS and then suspended in an RIPA buffer (50 mM Tris-HCl at pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl), followed by inversion mixing at 4° C. for eight hours for lysis. Nicastrin was immunoprecipitated (IP) from the resultant lysate fraction by use of an anti-nicastrin C-terminal antibody (N1660, Sigma), and the thus-precipitated nicastrin fraction was employed for the following analysis.
For Endo H or PNGase treatment, 200 mM citrate-NaOH (pH 5.8), 0.1% SDS, and 1% 2-mercaptoethanol were added to the nicastrin fraction, and the mixture was boiled at 95° C. for five minutes. 500 mU/mL Endoglycosidase H (Roche Applied Sciences) or 200 U/mL PNGase F (Roche Applied Sciences) was added to the mixture, and reaction was carried out at 37° C. overnight. Finally, a 5× sample buffer (1/4 amount of the reaction mixture) was added to the reaction mixture, and the resultant mixture was boiled at 95° C. for five minutes, whereby reaction was terminated.
For neuraminidase (sialidase) treatment, 50 mM Na-acetate (pH 5.2), 2 mM CaCl2, and 0.5% 2-mercaptoethanol were added to the nicastrin fraction, and the mixture was boiled at 95° C. for five minutes. 500 mU/mL Neuraminidase (Roche Applied Sciences) was added to the mixture, and reaction was carried out at 37° C. overnight. Finally, a 5× sample buffer (1/4 amount of the reaction mixture) was added to the reaction mixture, and the resultant mixture was boiled at 95° C. for five minutes, whereby reaction was terminated.
Samples prepared through treatment with the aforementioned deglycosylation enzymes were subjected to western blot analysis. As a result, PPMX0401, PPMX0408, and PPMX0410 were found to exhibit cross-reactivity to deglycosylated nicastrin (FIG. 4). In FIG. 4, "O" represents Endo H-resistant nicastrin; "black dot" represents completely deglycosylated nicastrin; and "Δ" represents neuraminidase-desialylated nicastrin.
These data (in particular, the fact that each of the above-prepared antibodies recognized nicastrin which had been completely deglycosylated by PNGase F) suggest that the antibody binds to nicastrin by recognizing a peptide chain of the protein rather than a sugar chain thereof.
Immunoprecipitation (IP) of Endogenous Nicastrin by Use of Anti-Nicastrin Antibody
HeLa cells were suspended in a cell homogenization buffer (10% glycerol-containing HEPES buffer (10 mM HEPES pH 7.4, 150 mM NaCl complete inhibitor cocktail (Roche Applied Sciences))) and homogenized by means of a homogenizer, followed by centrifugation at 1,500×g for 10 minutes. Subsequently, the resultant supernatant was centrifuged at 100,000×g for one hour, and the precipitate was employed as a HeLa cell membrane fraction. The cell membrane fraction was lysed in a 1% CHAPSO-containing HEPES buffer, to thereby yield a HeLa cell membrane lysate fraction. After IP of nicastrin from the lysate fraction by use of each of the above-prepared anti-nicastrin monoclonal antibodies, western blot analysis was carried out by use of various antibodies.
As a result, the above-prepared antibodies were found to be classified into two groups; i.e., antibodies which allow IP of only immature nicastrin (PPMX0401, PPMX0402, PPMX0407, and PPMX0409) (first group); and antibodies which allow IP of both immature nicastrin and mature nicastrin (PPMX0406, PPMX0408, and PPMX0410) (second group) (FIG. 5). In the case of the antibodies of the second group (PPMX0406, PPMX0408, and PPMX0410), presenilin, PEN-2, and APH-1aL, which are components of the γ-secretase complex, were coprecipitated, whereas in the case of the antibodies of the first group (PPMX0401, PPMX0402, PPMX0407, and PPMX0409), only APH-1aL was precipitated in a small amount. As has been reported, immature nicastrin binds to APH-1 and forms a sub-complex before it forms a γ-secretase complex (M. LaVoie, et al., J. Biol. Chem. 278 (2003) 37213-37222). Therefore, conceivably, each of the antibodies of the first group (PPMX0401, PPMX0402, PPMX0407, and PPMX0409) binds specifically to immature nicastrin contained in a nicastrin-APH-1 sub-complex.
As has also been reported, the structure of the extracellular domain of nicastrin changes with formation of the γ-secretase complex (K. Shirotani, et al., J. Biol. Chem. 278 (2003) 16474-16477). When the HeLa cell 1% CHAPSO lysate was treated with trypsin, the extracellular domain of nicastrin exhibited resistance to trypsin. Therefore, conceivably, the extracellular domain of nicastrin maintains its structure in the γ-secretase complex in the presence of 1% CHAPSO (FIG. 6). Thus, the data of the IP experiment suggest that the epitope site of each antibody of the first group is masked through structural change of nicastrin, whereas the epitope site of each antibody of the second group may be exposed even after structural change of nicastrin.
Immunostaining of Cultured Cells by Use of Anti-Nicastrin Monoclonal Antibody
Biochemical studies have reported that active γ-secretase containing mature nicastrin is localized to lipid rafts (Urano Y., Hayashi I., Isoo N., et al.: Association of active γ-secretase complex with lipid rafts. J. Lipid Res. 2005, 46: 904). In this Example, cultured cells were immunostained by use of the above-prepared antibodies, to thereby examine intracellular localization of nicastrin recognized by the antibodies. Intracellular localization of nicastrin was examined by use of HeLa cells and NKO cells. Cells were bonded, at an appropriate cell density, to a cover glass which had been coated with poly-D-lysine in advance, and the cells were washed with PBS and then fixed with PBS containing 4% paraformaldehyde. PBS containing 3% BSA was employed for blocking, and, in the case of permeation, Triton X-100 (final concentration: 0.1%) was further added. Each of the antibodies was diluted to an appropriate concentration with a blocking solution, and the thus-diluted antibody was reacted with the cells (at room temperature for three hours, or at 4° C. overnight). An anti-mouse or anti-rabbit immunoglobulin antibody bound to Alexa 488 or 546 was employed as a secondary antibody.
As a result, in HeLa cells, a granular structure and the cell membrane were stained in the presence of PPMX0408, whereas such a structure was not stained in the presence of PPMX0401 (FIG. 7). In NKO cells, a granular structure was not stained in the presence of PPMX0408. However, when wild-type nicastrin was introduced into NKO cells, there was obtained a stained image similar to that obtained in the case of HeLa cells (FIG. 8). These data indicate that the granular structure stained in the presence of PPMX0408 is derived from nicastrin.
Subsequently, in order to examine intracellular localization of the granular structure, co-staining was carried out by use of PPMX0408 and antibodies to various marker proteins. As a result, localization of the granular structure did not correspond to that of calnexin and giantin, which are marker proteins for endoplasmic reticulum and Golgi body, respectively (FIG. 9). In contrast, the results of staining in the presence of cholera toxin subunit B (CTB), which is used for staining of GM1 ganglioside present in lipid rafts, corresponded well to those of staining of the granular structure in the presence of PPMX0408. The results of staining in the presence of PPMX0401 did not correspond to those of staining in the presence of CTB (FIG. 10). Correspondence of localization similar to that described above was observed even under non-permeating conditions (i.e., no treatment with Triton X-100 during blocking) (FIG. 10). These data suggest that PPMX0408 recognizes mature nicastrin which is localized to lipid rafts (including cell membrane).
Neutralization of Human Active γ-Secretase Activity by Use of Anti-Nicastrin Monoclonal Antibody
Since PPMX0408 or PPMX0410 binds to mature nicastrin contained in active γ-secretase under the conditions where the γ-secretase complex is maintained, these antibodies are considered to affect γ-secretase activity. Therefore, a microsomal fraction of HeLa cells was lysed with 1% CHAPSO; each of the antibodies was added to an in vitro γ-secretase assay system employing an artificial substrate; and γ-secretase activity was determined on the basis of accumulation of de novo synthesized Aβ (Takasugi N., Tomita T., Hayashi I., Tsuruoka M., Niimura M., Takahashi Y., Thinakaran G., Iwatsubo T.: The role of presenilin cofactors in the γ-secretase complex. Nature 2003, 422: 438; and Takahashi Y., Hayashi I., Tominari Y., et al.: Sulindac sulfide is a non-competitive γ-secretase inhibitor that preferentially reduces Aβ 42 generation. J. Biol. Chem. 2003, 278: 18664).
When PPMX0401 was added (final concentration: 10 μg/mL), γ-secretase activity was maintained at almost the same level as in the case where PBS was added. In contrast, when PPMX0408 or PPMX0410 was added (final concentration: 10 μg/mL), γ-secretase activity was inhibited by about 20%, as compared with the case where PBS was added (FIG. 11). This suggest that the antibodies which bind to mature nicastrin exhibit γ-secretase inhibitory activity.
Effect of Anti-Nicastrin Monoclonal Antibody on Viability of Cancer Cell Lines
Firstly, in order to identify a cancer cell line exhibiting Notch-signaling-dependent survival, the effect of a γ-secretase inhibitor DAPT (H F. Dovey, et al., J. Neurochem. 76 (2001) 173-181) on survival of HeLa cells or A549 cells was evaluated through the MTT method. HeLa cells or A549 cells (5×103 cells) were inoculated onto a 96-well multiplate and treated with DAPT (final concentration: 100 μM) for 72 hours. After the 72-hour treatment, MTT diluted with PBS was added to the plate so that the final MTT concentration was 500 μg/mL, followed by culturing at 37° C. for three to four hours. Thereafter, stop solution (10% SDS, 0.01 M HCl) was added to the plate for termination of reaction, and the plate was allowed to stand still at 37° C. overnight, followed by dissolution of produced formazan. The formazan solution was uniformly mixed through pipetting, and absorbance was measured at 550 nm, to thereby calculate cell viability. As a result, the viability of DAPT-treated A549 cells was significantly lower than that of DAPT-untreated A549 cells. In contrast, no significant difference was observed in viability between DAPT-treated HeLa cells and DAPT-untreated HeLa cells (FIG. 12). Subsequently, in order to confirm that this reduction in cell viability was attributed to inhibition of γ-secretase activity, endogenous nicastrin of A549 cells was knocked down through treatment with nicastrin-corresponding short interference RNA (siRNA), and change in cell viability was determined. As a result, in the case of treatment with nicastrin siRNA, cell viability was reduced by about 20%, as compared with the case of treatment with siRNA having a random sequence (scramble) (FIG. 13). Under the nicastrin siRNA treatment conditions, expression of endogenous nicastrin was completely inhibited (FIG. 14). These data suggest that, unlike the case of HeLa cells, survival of A549 cells requires γ-secretase activity.
Subsequently, the effect of the above-prepared antibodies on survival of A549 cells was examined. Each of the antibodies was added to A549 cells so that the final antibody concentration was 10 μg/mL, and, 96 hours after addition of the antibody, cell viability was determined through the MTT method. As a result, the viability of PPMX0410-treated A549 cells was significantly lower than that of antibody-untreated A549 cells or PPMX0401-treated A549 cells (FIG. 15). These data suggest that an antibody exhibiting γ-secretase inhibitory activity has an ability to inhibit proliferation of cancer cells exhibiting γ-secretase-dependent survival.
Effect of Anti-Nicastrin Monoclonal Antibody on Proliferation of Leukemia Cell Lines
As has been reported, proliferation of cells of the following cell lines: TALL-1, ALL-SIL, and DND-41--which are isolated and established from patients with T-cell acute lymphoblastic leukemia (T-ALL)--requires Notch signaling (Weng, A. P., Ferrando, A. A., Lee, W., Morris, J. P. t., Silverman, L. B., Sanchez-Irizarry, C., Blacklow, S. C., Look, A. T. and Aster, J. C. (2004), Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306, 269-271). As has also been reported, in TALL-1 cells, somatic mutation is not found in the Notch1 gene, but in ALL-SIL cells or DND-41 cells, missense mutation occurs in the region (HDN) involved in interaction between an extracellular domain of Notch1 and TMIC (transmembrane-intracellular domain of Notch), and deletion (by mutation) occurs in the PEST region involved in degradation of NICD (Notch intracellular domain) (FIG. 16). Conceivably, mutation of the HDN region causes ligand-independent heterodimeric dissociation, shedding, and cleavage by γ-secretase, and deletion in the PEST region increases the stability of NICD, which induces abnormal activation of Notch signaling, thereby causing T-ALL.
Firstly, there was examined the effect of treatment of TALL-1, ALL-SIL, or DND-41 cells with a γ-secretase inhibitor on metabolism of Notch1. Through western blot analysis by use of an antibody mN1A to the intracellular ankyrin repeat domain of Notch1 (Chemicon, Cat #MAB5352), a band considered to be attributed to Notch1 TMIC was observed in the cases of all these types of cells. In the case of ALL-SIL cells or DND-41 cells, a band considered to be attributed to NEXT (Notch extracellular truncation) was observed at a position slightly below the TMIC band, and also a somewhat unclear band considered to be attributed to NICD was observed at a position below the NEXT band (FIG. 17). In the case of ALL-SIL cells or DND-41 cells, constitutive expression of NICD was determined by an antibody Val1744 (Cell Signaling, Cat #2421) specific to the cleaved N-terminal of NICD, but in the case of TALL-1 cells, expression of NICD was not observed. Subsequently, a γ-secretase inhibitor YO (concentration: 10, 100, or 1,000 nM) was added to the culture supernatant of each type of cells, and the cells were recovered 48 hours after addition of YO, followed by western blot analysis of the resultant lysate. As a result, in the case of YO treatment of ALL-SIL cells or DND-41 cells, NICD was found to disappear, and TMIC and NEXT were found to be accumulated (FIG. 17). These data suggest that Notch signaling is constitutively activated in at least both ALL-SIL cells and DND-41 cells.
Subsequently, the effect of YO treatment on proliferation of these cells was examined. Cells were inoculated onto a 96-well plate (5×103 cells/well) and cultured at 37° C. overnight. Then, a γ-secretase inhibitor YO was added to the plate, followed by culturing for seven days. Thereafter, percent cell proliferation was determined by use of Alamar Blue (Serotec). Alamar Blue was added to the culture liquid in an amount of 1/10 that of the culture liquid, followed by culturing at 37° C. for four hours. Subsequently, the resultant culture supernatant was recovered. Fluorescence in the culture supernatant was measured by means of a plate reader (excitation wavelength: 530 nm, fluorescence wavelength: 590 nm), and percent cell proliferation was calculated by use of the following formula.
cell proliferation ( % ) = A 590 PC 590 × 100 ##EQU00001##
In the above formula, "A590" represents the absorbance of a sample at 590 nm, and "PC590" represents the absorbance of a positive control group (treated with PBS or DMSO) at 590 nm. As a result, proliferation of TALL-1 cells or DND-41 cells was found to be inhibited through YO treatment. Specifically, through treatment with 10 nM YO, proliferation of TALL-1 cells or DND-41 cells was inhibited by about 60% or about 50%, respectively, and, through treatment with 1,000 nM YO, proliferation of TALL-1 cells or DND-41 cells was inhibited by about 80% (FIG. 18). Unexpectedly, virtually no inhibition of cell proliferation was observed in ALL-SIL cells, in which NICD was found to disappear through YO treatment (as determined by western blot analysis). These data indicate that TALL-1 cells or DND-41 cells exhibit γ-secretase activity-dependent proliferation.
The above-obtained data suggest that, among the examined T-ALL-derived cells, at least DND-41 cells exhibit Notch signaling/γ-secretase activity-dependent proliferation. Therefore, the effect of PPMX0410 (i.e., an anti-nicastrin antibody) on proliferation of DND-41 cells was examined. PPMX0410 or a mouse IgG fraction (concentration: 0.1, 1, 10, or 100 μg/mL) was added to a DND-41 cell culture supernatant, followed by culturing for seven days. Thereafter, percent cell proliferation was determined by use of Alamar Blue. As a result, percent cell proliferation tended to slightly increase in an IgG-fraction-concentration-dependent manner, but tended to lower through addition of PPMX0410. Specifically, proliferation of DND-41 cells was inhibited by about 60% through addition of 100 μg/mL PPMX0410 (FIG. 19). These data indicate that PPMX0410 inhibits Notch signaling/γ-secretase activity-dependent proliferation of T-ALL cells.
On the basis of these results, PPMX0410 was deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Central 6th, Tsukuba Center, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan, Postal Code 305-8566) (deposition date: Apr. 21, 2006, accession number: FERM-AP20895).
Effect of Anti-Nicastrin Monoclonal Antibody in Inhibiting Binding Between Nicastrin and Substrate
As has been reported, nicastrin may function as a substrate receptor in the γ-secretase complex (Shah S., Lee S F., Tabuchi K., Hao Y H., Yu C., LaPlant Q., Ball H., Dann C E 3rd, Sudhof T., Yu G.: Nicastrin functions as a γ-secretase-substrate receptor. Cell 2005, 122: 435).
Therefore, there was examined a possibility that PPMX0410 exhibits γ-secretase inhibitory activity by inhibiting interaction between γ-secretase and a substrate therefor.
Firstly, there was expressed, in Sf9 cells, nicastrin (having a V5-His tag sequence added at the carboxyl terminus) or N100-FLAG (100 amino acid residues (No. 1711 to No. 1809) of Notch receptor including the intramembrane sequence (NH2-MVKSEPVEPPLPSQLHLVYVAAAAFVLLFFVGCGVLLSRKRRRQHGQLWFPEGFKVSEASK KKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLE-COOH) and having a FLAG-His tag (DYKDDDDKGSHHHHHH) added at the carboxyl terminus) (SEQ ID NOs: 13 and 14), Lee S F., Shah S., Li H., Yu C., Han W., Yu G.: Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid-β precursor protein and Notch. J. Biol. Chem. 2002 277: 45013). Subsequently, a cell membrane fraction was prepared through the method described above in Example 9.
The resultant cell fraction was lysed in a HEPES buffer containing 1% CHAPSO, to thereby yield a nicastrin fraction or an N100 fraction.
The nicastrin fraction was mixed with PPMX0401 or PPMX0410 diluted to an appropriate concentration with PBS, and reaction was carried out at 4° C. overnight. Thereafter, the N100 fraction was added to the reaction mixture, followed by inversion mixing for three hours. A 1% CHAPSO-containing HEPES buffer was employed during mixing of the nicastrin fraction with the antibody, and a 0.5% CHAPSO-containing HEPES buffer was employed after addition of the N100 fraction.
Nicastrin and N100-FLAG were coprecipitated from the resultant nicastrin-N100 fraction mixture by use of anti-V5-antibody-bound V5-agarose beads (SIGMA) or anti-FLAG-antibody-bound M2-agarose beads (SIGMA), and the precipitated fraction was subjected to western blot analysis by use of an anti-V5 antibody (FIG. 20).
In the case where a nicastrin fraction which had been denatured with 0.1% SDS in advance was employed, the amount of nicastrin precipitated by the M2-agarose beads (i.e., nicastrin bound to N100-FLAG) was reduced, as compared with the case where a native nicastrin fraction was employed (comparison between lanes "D" and "N" in FIG. 20).
Thus, this experiment system was considered to be applicable to detection of structure-dependent binding of nicastrin to N100.
Under the aforementioned conditions, PPMX0401 or PPMX0410 was added, and the amount of nicastrin precipitated by the M2-agarose beads was measured, followed by comparison of the resultant measurement data. In the case where PPMX0410 was added at a concentration of 10 or 100 μg/mL, the amount of nicastrin precipitated was found to be considerably reduced (FIG. 21). When the measurement data were normalized with the amount of nicastrin precipitated by the V5-agarose beads, PPMX0410 (at the aforementioned concentrations) was found to inhibit binding between nicastrin and N100-FLAG by about 60% (FIG. 21).
These data suggest that PPMX0410 inhibits γ-secretase activity by inhibiting binding between nicastrin and a substrate for the enzyme. Thus, these data suggest that an antibody exhibiting potent γ-secretase inhibitory activity can be selected on the basis of inhibition of binding between nicastrin and a substrate for the enzyme.
Inhibition of γ-Secretase Activity by Anti-Nicastrin Antibody in Living Cells
An experiment was carried out by means of a GAL4-UAS system employing reporter cells, in order to determine whether or not PPMX0410--which inhibits γ-secretase activity in an in vitro reaction system--also inhibits cleavage mediated by γ-secretase activity in living cells.
C99 is a fragment produced through cleavage of APP (amyloid precursor protein) by BACE (β-site APP cleaving enzyme) and serves as a direct substrate for γ-secretase.
A preproenkephalin-derived signal peptide was inserted into C99, and GAL4 (i.e., a yeast-derived transcription factor) was inserted immediately downstream of the transmembrance domain of C99, to thereby prepare a construct (SC100G). The construct (SC100G) was subcloned into pcDNA3.1/Hygro vector (Invitrogen).
GAL4/VP16 was bound to NΔE (including N99), in which deletion occurs in the extracellular domain of Notch receptor and which serves as a direct substrate for γ-secretase in a ligand-independent manner, to thereby prepare a construct (NΔEGV, Taniguchi Y., Karlstrom H., Lundkvist J., Mizutani T., Otaka A., Vestling M., Bernstein A., Donoviel D., Lendahl U., Honjo T.: Notch receptor cleavage depends on but is not directly executed by presenilins. Proc. Natl. Acad. Sci. U.S.A 2002, 99: 4014). The construct (NΔEGV) was subcloned into pcDNA3.1 vector (pcDNA3.1-NΔEGV).
A UAS sequence was inserted upstream of luciferase in pGL3 (R2.2) vector (Promega), to thereby prepare a construct (UAS-luc), and the construct was employed as a reporter construct. eGFP was subcloned into pcDNA3 (Invitrogen), and the thus-prepared pcDNA3-eGFP was employed as a control vector for monitoring the number of cells. HEK293 cells were transfected with pcDNA3.1-SC100G and UAS-luc, or transfected with pcDNA3.1-NΔEGV, UAS-luc, and pcDNA3-eGFP by use of Lipofectamine 2000 (Invitrogen). Cells constitutively expressing nicastrin (HEK/SC100G cells or HEK/NΔEGV cells) were selected by use of an antibiotic-resistant marker (Hygromycin (Wako Pure Chemical Industries, Ltd.) or G418 (CALBIOCHEM), respectively).
HEK/SC100G cells or HEK/NΔEGV cells were inoculated into a 48-well multiplate (2.5×104 cells). After culturing at 37° C. for 24 hours, PBS or PPMX0410 diluted to an appropriate concentration with PBS was added to the plate. Cells treated with DMSO or DAPT (final concentration: 10 μM) (i.e., γ-secretase-activity-inhibiting control) were also provided. After culturing at 37° C. for 36 hours, n-butyric acid (final concentration: 10 mM) was added for induction of transgene expression. After culturing for 12 hours, cells and a culture supernatant were recovered, and the amount of Aβ was determined through a reporter assay or ELISA.
The recovered cells were lysed in a lysis buffer (Promega), and the resultant lysate was subjected to the reporter assay. PicaGene (Toyo Ink Mfg. Co., Ltd.) was employed as a luminescent substrate. The amount of luciferase luminescence was normalized by the concentration of protein (in the case of HEK/SC100G cells), or by the amount of eGFP luminescence (in the case of HEK/NΔEGV cells), to thereby yield relative light unit (RLU). The amount of Aβ secreted in the culture supernatant of HEK/SC100G cells was determined through ELISA, and the thus-determined Aβ amount was normalized by the concentration of protein similar to the case of normalization of the amount of luciferase luminescence.
As a result, PPMX0410 was found to inhibit, in a concentration-dependent manner, reporter activity (FIG. 22A) and Aβ secretion (FIG. 22B) in HEK/SC100G cells, and reporter activity (FIG. 22C) in HEK/NΔEGV cells. Under the aforementioned conditions, DAPT (i.e., a γ-secretase inhibitor) was found to inhibit reporter activity in both HEK/SC100G cells and HEK/NΔEGV cells.
These data indicate that PPMX0410 also inhibits γ-secretase activity in living cells and inhibits intramembrane protein cleavage in APP or Notch receptor.
The method described in Example 14 can be employed for high throughput screening. Therefore, the method is considered applicable to selection of an antibody exhibiting potent γ-secretase inhibitory activity.
1912944DNAHomo sapiens 1cgcctggaaa cacgaacttc cggtctctta ggctccgggc cacagagacg gtgtcagtgg 60tagcctagag aggccgctaa cagacaggag ccgaacgggg gcttccgctc agcagagagg 120caagatggct acggcagggg gtggctctgg ggctgacccg ggaagtcggg gtctccttcg 180ccttctgtct ttctgcgtcc tactagcagg tttgtgcagg ggaaactcag tggagaggaa 240gatatatatc cccttaaata aaacagctcc ctgtgttcgc ctgctcaacg ccactcatca 300gattggctgc cagtcttcaa ttagtggaga cacaggggtt atccacgtag tagagaaaga 360ggaggaccta cagtgggtat tgactgatgg ccccaacccc ccttacatgg ttctgctgga 420gagcaagcat tttaccaggg atttaatgga gaagctgaaa gggagaacca gccgaattgc 480tggtcttgca gtgtccttga ccaagcccag tcctgcctca ggcttctctc ctagtgtaca 540gtgcccaaat gatgggtttg gtgtttactc caattcctat gggccagagt ttgctcactg 600cagagaaata cagtggaatt cgctgggcaa tggtttggct tatgaagact ttagtttccc 660catctttctt cttgaagatg aaaatgaaac caaagtcatc aagcagtgct atcaagatca 720caacctgagt cagaatggct cagcaccaac cttcccacta tgtgccatgc agctcttttc 780acacatgcat gctgtcatca gcactgccac ctgcatgcgg cgcagctcca tccaaagcac 840cttcagcatc aacccagaaa tcgtctgtga ccccctgtct gattacaatg tgtggagcat 900gctaaagcct ataaatacaa ctgggacatt aaagcctgac gacagggttg tggttgctgc 960cacccggctg gatagtcgtt cctttttctg gaatgtggcc ccaggggctg aaagcgcagt 1020ggcttccttt gtcacccagc tggctgctgc tgaagctttg caaaaggcac ctgatgtgac 1080caccctgccc cgcaatgtca tgtttgtctt ctttcaaggg gaaacttttg actacattgg 1140cagctcgagg atggtctacg atatggagaa gggcaagttt cccgtgcagt tagagaatgt 1200tgactcattt gtggagctgg gacaggtggc cttaagaact tcattagagc tttggatgca 1260cacagatcct gtttctcaga aaaatgagtc tgtacggaac caggtggagg atctcctggc 1320cacattggag aagagtggtg ctggtgtccc tgctgtcatc ctcaggaggc caaatcagtc 1380ccagcctctc ccaccatctt ccctgcagcg atttcttcga gctcgaaaca tctctggcgt 1440tgttctggct gaccactctg gtgccttcca taacaaatat taccagagta tttacgacac 1500tgctgagaac attaatgtga gctatcccga atggctgagc cctgaagagg acctgaactt 1560tgtaacagac actgccaagg ccctggcaga tgtggccacg gtgctgggac gtgctctgta 1620tgagcttgca ggaggaacca acttcagcga cacagttcag gctgatcccc aaacggttac 1680ccgcctgctc tatgggttcc tgattaaagc caacaactca tggttccagt ctatcctcag 1740gcaggaccta aggtcctact tgggtgacgg gcctcttcaa cattacatcg ctgtctccag 1800ccccaccaac accacttatg ttgtacagta tgccttggca aatttgactg gcacagtggt 1860caacctcacc cgagagcagt gccaggatcc aagtaaagtc ccaagtgaaa acaaggatct 1920gtatgagtac tcatgggtcc agggcccttt gcattctaat gagacggacc gactcccccg 1980gtgtgtgcgt tctactgcac gattagccag ggccttgtct cctgcctttg aactgagtca 2040gtggagctct actgaatact ctacatggac tgagagccgc tggaaagata tccgtgcccg 2100gatatttctc atcgccagca aagagcttga gttgatcacc ctgacagtgg gcttcggcat 2160cctcatcttc tccctcatcg tcacctactg catcaatgcc aaagctgatg tccttttcat 2220tgctccccgg gagccaggag ctgtgtcata ctgaggagga ccccagcttt tcttgccagc 2280tcagcagttc acttcctaga gcatctgtcc cactgggaca caaccactaa tttgtcactg 2340gaacctccct gggcctgtct cagattggga ttaacataaa agagtggaac tatccaaaag 2400agacagggag aaataaataa attgcctccc ttcctccgct cccctttccc atcacccctt 2460ccccatttcc tcttccttct ctactcatgc cagattttgg gattacaaat agaagcttct 2520tgctcctgtt taactcccta gttacccacc ctaatttgcc cttcaggacc cttctacttt 2580ttccttcctg ccctgtacct ctctctgctc ctcaccccca cccctgtacc cagccacctt 2640cctgactggg aaggacataa aaggtttaat gtcagggtca aactacattg agcccctgag 2700gacaggggca tctctgggct gagcctactg tctccttccc actgtccttt ctccaggccc 2760tcagatggca cattagggtg ggcgtgctgc gggtgggtat cccacctcca gcccacagtg 2820ctcagttgta ctttttatta agctgtaata tctatttttg tttttgtctt tttcctttat 2880tctttttgta aatatatata taatgagttt cattaaaata gattatccca cacgaaaaaa 2940aaaa 29442709PRTHomo sapiens 2Met Ala Thr Ala Gly Gly Gly Ser Gly Ala Asp Pro Gly Ser Arg Gly1 5 10 15Leu Leu Arg Leu Leu Ser Phe Cys Val Leu Leu Ala Gly Leu Cys Arg 20 25 30Gly Asn Ser Val Glu Arg Lys Ile Tyr Ile Pro Leu Asn Lys Thr Ala 35 40 45Pro Cys Val Arg Leu Leu Asn Ala Thr His Gln Ile Gly Cys Gln Ser 50 55 60Ser Ile Ser Gly Asp Thr Gly Val Ile His Val Val Glu Lys Glu Glu65 70 75 80Asp Leu Gln Trp Val Leu Thr Asp Gly Pro Asn Pro Pro Tyr Met Val 85 90 95Leu Leu Glu Ser Lys His Phe Thr Arg Asp Leu Met Glu Lys Leu Lys 100 105 110Gly Arg Thr Ser Arg Ile Ala Gly Leu Ala Val Ser Leu Thr Lys Pro 115 120 125Ser Pro Ala Ser Gly Phe Ser Pro Ser Val Gln Cys Pro Asn Asp Gly 130 135 140Phe Gly Val Tyr Ser Asn Ser Tyr Gly Pro Glu Phe Ala His Cys Arg145 150 155 160Glu Ile Gln Trp Asn Ser Leu Gly Asn Gly Leu Ala Tyr Glu Asp Phe 165 170 175Ser Phe Pro Ile Phe Leu Leu Glu Asp Glu Asn Glu Thr Lys Val Ile 180 185 190Lys Gln Cys Tyr Gln Asp His Asn Leu Ser Gln Asn Gly Ser Ala Pro 195 200 205Thr Phe Pro Leu Cys Ala Met Gln Leu Phe Ser His Met His Ala Val 210 215 220Ile Ser Thr Ala Thr Cys Met Arg Arg Ser Ser Ile Gln Ser Thr Phe225 230 235 240Ser Ile Asn Pro Glu Ile Val Cys Asp Pro Leu Ser Asp Tyr Asn Val 245 250 255Trp Ser Met Leu Lys Pro Ile Asn Thr Thr Gly Thr Leu Lys Pro Asp 260 265 270Asp Arg Val Val Val Ala Ala Thr Arg Leu Asp Ser Arg Ser Phe Phe 275 280 285Trp Asn Val Ala Pro Gly Ala Glu Ser Ala Val Ala Ser Phe Val Thr 290 295 300Gln Leu Ala Ala Ala Glu Ala Leu Gln Lys Ala Pro Asp Val Thr Thr305 310 315 320Leu Pro Arg Asn Val Met Phe Val Phe Phe Gln Gly Glu Thr Phe Asp 325 330 335Tyr Ile Gly Ser Ser Arg Met Val Tyr Asp Met Glu Lys Gly Lys Phe 340 345 350Pro Val Gln Leu Glu Asn Val Asp Ser Phe Val Glu Leu Gly Gln Val 355 360 365Ala Leu Arg Thr Ser Leu Glu Leu Trp Met His Thr Asp Pro Val Ser 370 375 380Gln Lys Asn Glu Ser Val Arg Asn Gln Val Glu Asp Leu Leu Ala Thr385 390 395 400Leu Glu Lys Ser Gly Ala Gly Val Pro Ala Val Ile Leu Arg Arg Pro 405 410 415Asn Gln Ser Gln Pro Leu Pro Pro Ser Ser Leu Gln Arg Phe Leu Arg 420 425 430Ala Arg Asn Ile Ser Gly Val Val Leu Ala Asp His Ser Gly Ala Phe 435 440 445His Asn Lys Tyr Tyr Gln Ser Ile Tyr Asp Thr Ala Glu Asn Ile Asn 450 455 460Val Ser Tyr Pro Glu Trp Leu Ser Pro Glu Glu Asp Leu Asn Phe Val465 470 475 480Thr Asp Thr Ala Lys Ala Leu Ala Asp Val Ala Thr Val Leu Gly Arg 485 490 495Ala Leu Tyr Glu Leu Ala Gly Gly Thr Asn Phe Ser Asp Thr Val Gln 500 505 510Ala Asp Pro Gln Thr Val Thr Arg Leu Leu Tyr Gly Phe Leu Ile Lys 515 520 525Ala Asn Asn Ser Trp Phe Gln Ser Ile Leu Arg Gln Asp Leu Arg Ser 530 535 540Tyr Leu Gly Asp Gly Pro Leu Gln His Tyr Ile Ala Val Ser Ser Pro545 550 555 560Thr Asn Thr Thr Tyr Val Val Gln Tyr Ala Leu Ala Asn Leu Thr Gly 565 570 575Thr Val Val Asn Leu Thr Arg Glu Gln Cys Gln Asp Pro Ser Lys Val 580 585 590Pro Ser Glu Asn Lys Asp Leu Tyr Glu Tyr Ser Trp Val Gln Gly Pro 595 600 605Leu His Ser Asn Glu Thr Asp Arg Leu Pro Arg Cys Val Arg Ser Thr 610 615 620Ala Arg Leu Ala Arg Ala Leu Ser Pro Ala Phe Glu Leu Ser Gln Trp625 630 635 640Ser Ser Thr Glu Tyr Ser Thr Trp Thr Glu Ser Arg Trp Lys Asp Ile 645 650 655Arg Ala Arg Ile Phe Leu Ile Ala Ser Lys Glu Leu Glu Leu Ile Thr 660 665 670Leu Thr Val Gly Phe Gly Ile Leu Ile Phe Ser Leu Ile Val Thr Tyr 675 680 685Cys Ile Asn Ala Lys Ala Asp Val Leu Phe Ile Ala Pro Arg Glu Pro 690 695 700Gly Ala Val Ser Tyr70538064DNAMus musculusCDS(79)..(7671) 3gtggtgtgcg tcaacgtccg atccccgccg gccaccccaa gaggccgccg ccgggctgcg 60ggcagctggc gagcaggc atg cca cgg ctc ctg acg ccc ctt ctc tgc cta 111Met Pro Arg Leu Leu Thr Pro Leu Leu Cys Leu1 5 10acg ctg ctg ccc gcg cgc gcc gca aga ggc ttg aga tgc tcc cag cca 159Thr Leu Leu Pro Ala Arg Ala Ala Arg Gly Leu Arg Cys Ser Gln Pro 15 20 25agt ggg acc tgc ctg aat gga ggt agg tgc gaa gtg gcc agc ggc act 207Ser Gly Thr Cys Leu Asn Gly Gly Arg Cys Glu Val Ala Ser Gly Thr 30 35 40gaa gcc tgt gtt gcc agc ggc agc ttt gtg ggc caa cga tgc cag gac 255Glu Ala Cys Val Ala Ser Gly Ser Phe Val Gly Gln Arg Cys Gln Asp 45 50 55ccc aat cct tgc ctc agc aca cgg tgt aag aat gct gga acg tgc tat 303Pro Asn Pro Cys Leu Ser Thr Arg Cys Lys Asn Ala Gly Thr Cys Tyr60 65 70 75gtt gtg gac cat ggt ggc atc gtg gac tat gcc tgc agc tgt ccc ctg 351Val Val Asp His Gly Gly Ile Val Asp Tyr Ala Cys Ser Cys Pro Leu 80 85 90ggt ttc tct ggg ccc ctc tgc ctg aca cct ctg gac aag ccc tgc ctg 399Gly Phe Ser Gly Pro Leu Cys Leu Thr Pro Leu Asp Lys Pro Cys Leu 95 100 105gcc aac ccc tgc cgc aat ggg ggc acc tgt gac ctg ctc act ctc aca 447Ala Asn Pro Cys Arg Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr 110 115 120gag tac aag tgc cgc tgc tct cca ggg tgg tca gga aaa tca tgt cag 495Glu Tyr Lys Cys Arg Cys Ser Pro Gly Trp Ser Gly Lys Ser Cys Gln 125 130 135cag gct gac ccc tgt gcc tcc aac ccc tgt gcc aat ggt ggc cag tgc 543Gln Ala Asp Pro Cys Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys140 145 150 155ctg ccc ttt gag tct tca tac atc tgt cgc tgc ccg cct ggc ttc cat 591Leu Pro Phe Glu Ser Ser Tyr Ile Cys Arg Cys Pro Pro Gly Phe His 160 165 170ggc ccc acc tgc agg caa gat gtt aat gag tgc agc cag aac cct ggg 639Gly Pro Thr Cys Arg Gln Asp Val Asn Glu Cys Ser Gln Asn Pro Gly 175 180 185ctg tgc cgc cat gga ggc cac tgc cac aat gag atc ggc tcc tat cgc 687Leu Cys Arg His Gly Gly His Cys His Asn Glu Ile Gly Ser Tyr Arg 190 195 200tgt gcc tgc tgt gcc acc cat act ggt ccc cac tgt gaa ctg ccc tat 735Cys Ala Cys Cys Ala Thr His Thr Gly Pro His Cys Glu Leu Pro Tyr 205 210 215gtg ccc tgc agc ccc tca ccc tgc cag aat gga gca acc tgc cgt cct 783Val Pro Cys Ser Pro Ser Pro Cys Gln Asn Gly Ala Thr Cys Arg Pro220 225 230 235aca ggg gac acc acc cac gag tgt gcc tgc ttg cca ggt ttt gct gga 831Thr Gly Asp Thr Thr His Glu Cys Ala Cys Leu Pro Gly Phe Ala Gly 240 245 250cag aac tgt gaa gaa aat gtg gat gac tgt cca gga aac aac tgc aag 879Gln Asn Cys Glu Glu Asn Val Asp Asp Cys Pro Gly Asn Asn Cys Lys 255 260 265aat ggg ggt gcc tgt gtg gac ggc gtg aat acc tac aat tgc cgc tgc 927Asn Gly Gly Ala Cys Val Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys 270 275 280cca ccg gag gtg acg ggt cag tac tgt aca gag gat gtg gac gaa tgt 975Pro Pro Glu Val Thr Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys 285 290 295cag ctc atg cca aat gcc tgc cag aat gcg gga acc tgc cac aac aca 1023Gln Leu Met Pro Asn Ala Cys Gln Asn Ala Gly Thr Cys His Asn Thr300 305 310 315cac ggc ggc tac aac tgt gtg tgt gtc aat ggg tgg act ggc gag gac 1071His Gly Gly Tyr Asn Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp 320 325 330tgc agt gag aac att gat gac tgt gcc agt gcc gcc tgt ttc cag ggt 1119Cys Ser Glu Asn Ile Asp Asp Cys Ala Ser Ala Ala Cys Phe Gln Gly 335 340 345gcc act tgc cac gac cgt gtg gct tcc ttc tac tgc gaa tgt ccg cat 1167Ala Thr Cys His Asp Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His 350 355 360ggg cgc aca ggt ctg ctg tgc cac ctc aag cat gcg tgc atc agc aac 1215Gly Arg Thr Gly Leu Leu Cys His Leu Lys His Ala Cys Ile Ser Asn 365 370 375ccc tgc aac gag ggc tcc aac tgt gac acc aac cct gtc aac ggc aaa 1263Pro Cys Asn Glu Gly Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys380 385 390 395cga atc tgc acc tgc ccc tcg ggg tac aca ggg cca gcc tgc agc cag 1311Arg Ile Cys Thr Cys Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln 400 405 410gac gtg gat gag tgt gat ctg ggt gcc aac cgt tgt gag cac gca ggc 1359Asp Val Asp Glu Cys Asp Leu Gly Ala Asn Arg Cys Glu His Ala Gly 415 420 425aaa tgc ctc aac aca ctg ggt tct ttt gag tgc cag tgt cta cag ggc 1407Lys Cys Leu Asn Thr Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly 430 435 440tac acg gga ccc ggc tgt gag att gat gtt aat gag tgc atc tcc aac 1455Tyr Thr Gly Pro Gly Cys Glu Ile Asp Val Asn Glu Cys Ile Ser Asn 445 450 455cca tgt cag aat gac gcc act tgc ctg gac cag att ggg gag ttc caa 1503Pro Cys Gln Asn Asp Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln460 465 470 475tgc ata tgt atg cca ggt tat gaa ggt gta tac tgt gaa atc aac acg 1551Cys Ile Cys Met Pro Gly Tyr Glu Gly Val Tyr Cys Glu Ile Asn Thr 480 485 490gat gag tgc gcc agc agc ccc tgt ctg cac aat ggc cac tgc atg gac 1599Asp Glu Cys Ala Ser Ser Pro Cys Leu His Asn Gly His Cys Met Asp 495 500 505aag atc cat gag ttc caa tgt cag tgc ccc aaa ggc ttc aac ggg cac 1647Lys Ile His Glu Phe Gln Cys Gln Cys Pro Lys Gly Phe Asn Gly His 510 515 520ctg tgc cag tat gat gtg gat gag tgt gcc agc aca cca tgc aag aac 1695Leu Cys Gln Tyr Asp Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn 525 530 535ggt gcc aag tgc ctg gat ggg ccc aac acc tat acc tgc gtg tgt aca 1743Gly Ala Lys Cys Leu Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr540 545 550 555gaa ggt tac aca ggg acc cac tgc gaa gtg gac att gac gag tgt gac 1791Glu Gly Tyr Thr Gly Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp 560 565 570cct gac ccc tgc cac tat ggt tcc tgt aag gat ggt gtg gcc acc ttt 1839Pro Asp Pro Cys His Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe 575 580 585acc tgc ctg tgc cag cca ggc tac aca ggc cat cac tgt gag acc aac 1887Thr Cys Leu Cys Gln Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn 590 595 600atc aat gag tgc cac agc caa ccg tgc cgc cat ggg ggc acc tgc cag 1935Ile Asn Glu Cys His Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln 605 610 615gac cgt gac aac tcc tac ctc tgc tta tgc ctc aag gga acc aca ggg 1983Asp Arg Asp Asn Ser Tyr Leu Cys Leu Cys Leu Lys Gly Thr Thr Gly620 625 630 635ccc aac tgt gag atc aac ctg gat gac tgc gcc agc aac ccc tgt gac 2031Pro Asn Cys Glu Ile Asn Leu Asp Asp Cys Ala Ser Asn Pro Cys Asp 640 645 650tct ggc acc tgt ctg gac aag att gat ggc tac gaa tgt gcc tgt gaa 2079Ser Gly Thr Cys Leu Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu 655 660 665cca ggc tac aca gga agc atg tgt aac gtc aac att gac gaa tgt gcg 2127Pro Gly Tyr Thr Gly Ser Met Cys Asn Val Asn Ile Asp Glu Cys Ala 670 675 680ggc agc ccc tgc cac aac ggg ggc act tgt gag gat ggc atc gcg ggc 2175Gly Ser Pro Cys His Asn Gly Gly Thr Cys Glu Asp Gly Ile Ala Gly 685 690 695ttc act tgc cgc tgc ccc gag ggc tac cat gac ccc acg tgc ctg tcc 2223Phe Thr Cys Arg Cys Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser700 705 710 715gag gtc aac gag tgc aac agt aac ccc tgc atc cac gga gct tgc cgg 2271Glu Val Asn Glu Cys Asn Ser Asn Pro Cys Ile His Gly Ala Cys Arg 720 725 730gat ggc ctc aat ggg tac aag tgt gac tgt gcc cct ggg tgg agt gga 2319Asp Gly Leu Asn Gly Tyr Lys Cys Asp Cys Ala Pro Gly Trp Ser Gly 735 740 745aca aac tgt gac atc aac aac aac gag tgt gag tcc aac cct tgt gtc 2367Thr Asn Cys Asp Ile Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val 750 755 760aac ggt ggc acc tgc aag gac atg acc agt ggc tac gta tgc acc tgc 2415Asn Gly Gly Thr Cys Lys Asp Met Thr Ser Gly Tyr Val Cys Thr Cys 765 770 775cga gaa ggc ttc agt
ggc cct aat tgc cag acc aac atc aac gaa tgt 2463Arg Glu Gly Phe Ser Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys780 785 790 795gcc tcc aac ccc tgc ctg aac cag ggg acc tgc att gat gat gtc gct 2511Ala Ser Asn Pro Cys Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala 800 805 810gga tac aag tgc aac tgt cct ctg cca tat aca gga gcc acg tgt gag 2559Gly Tyr Lys Cys Asn Cys Pro Leu Pro Tyr Thr Gly Ala Thr Cys Glu 815 820 825gtg gtg ttg gcc cca tgt gct acc agc ccc tgc aaa aac agc ggc gta 2607Val Val Leu Ala Pro Cys Ala Thr Ser Pro Cys Lys Asn Ser Gly Val 830 835 840tgc aag gag tct gaa gac tat gag agt ttc tcc tgt gtc tgt ccc aca 2655Cys Lys Glu Ser Glu Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr 845 850 855ggc tgg caa ggt caa acc tgc gag gtt gac atc aat gag tgt gtg aaa 2703Gly Trp Gln Gly Gln Thr Cys Glu Val Asp Ile Asn Glu Cys Val Lys860 865 870 875agc cca tgt cgc cat ggg gcc tcc tgc cag aac acc aat ggc agc tac 2751Ser Pro Cys Arg His Gly Ala Ser Cys Gln Asn Thr Asn Gly Ser Tyr 880 885 890cgc tgc ctc tgc cag gcc ggc tat aca ggt cgc aac tgt gag agt gac 2799Arg Cys Leu Cys Gln Ala Gly Tyr Thr Gly Arg Asn Cys Glu Ser Asp 895 900 905atc gat gac tgc cgc ccc aac ccg tgt cac aat ggg ggt tcc tgc acc 2847Ile Asp Asp Cys Arg Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr 910 915 920gat ggc atc aac aca gcc ttc tgc gac tgc ctg ccc ggc ttc cag ggt 2895Asp Gly Ile Asn Thr Ala Phe Cys Asp Cys Leu Pro Gly Phe Gln Gly 925 930 935gcc ttc tgt gag gag gac atc aat gaa tgt gcc agc aat ccc tgc caa 2943Ala Phe Cys Glu Glu Asp Ile Asn Glu Cys Ala Ser Asn Pro Cys Gln940 945 950 955aat gga gcc aat tgc act gac tgt gtg gac agc tac aca tgt acc tgc 2991Asn Gly Ala Asn Cys Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys 960 965 970ccc gtg ggc ttc aat ggc atc cac tgc gag aac aac aca cct gac tgt 3039Pro Val Gly Phe Asn Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys 975 980 985act gag agc tcc tgc ttc aat ggt ggt acc tgt gtg gat ggt atc aac 3087Thr Glu Ser Ser Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn 990 995 1000tcc ttc acc tgt ctg tgt cca cct ggc ttc acg ggc agc tac tgt 3132Ser Phe Thr Cys Leu Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys 1005 1010 1015cag tat gat gtc aat gag tgt gat tca cgg ccc tgt ctg cac ggt 3177Gln Tyr Asp Val Asn Glu Cys Asp Ser Arg Pro Cys Leu His Gly 1020 1025 1030ggt acc tgc caa gac agc tat ggt act tat aag tgt acc tgc cca 3222Gly Thr Cys Gln Asp Ser Tyr Gly Thr Tyr Lys Cys Thr Cys Pro 1035 1040 1045cag ggc tac act ggt ctc aac tgc cag aac ctt gtg cgc tgg tgc 3267Gln Gly Tyr Thr Gly Leu Asn Cys Gln Asn Leu Val Arg Trp Cys 1050 1055 1060gac tcg gct ccc tgc aag aat ggt ggc agg tgc tgg cag acc aac 3312Asp Ser Ala Pro Cys Lys Asn Gly Gly Arg Cys Trp Gln Thr Asn 1065 1070 1075acg cag tac cac tgt gag tgc cgc agc ggc tgg act ggc gtc aac 3357Thr Gln Tyr His Cys Glu Cys Arg Ser Gly Trp Thr Gly Val Asn 1080 1085 1090tgc gac gtg ctc agt gtg tcc tgt gag gtg gct gca cag aag cga 3402Cys Asp Val Leu Ser Val Ser Cys Glu Val Ala Ala Gln Lys Arg 1095 1100 1105ggc att gac gtc act ctc ctg tgc cag cat gga ggg ctc tgt gtg 3447Gly Ile Asp Val Thr Leu Leu Cys Gln His Gly Gly Leu Cys Val 1110 1115 1120gat gag gga gat aaa cat tac tgc cac tgc cag gca ggc tac acg 3492Asp Glu Gly Asp Lys His Tyr Cys His Cys Gln Ala Gly Tyr Thr 1125 1130 1135ggc agc tac tgt gag gac gag gtg gac gag tgc tca cct aac ccc 3537Gly Ser Tyr Cys Glu Asp Glu Val Asp Glu Cys Ser Pro Asn Pro 1140 1145 1150tgc cag aat gga gct acc tgc act gac tat ctc ggc ggc ttt tcc 3582Cys Gln Asn Gly Ala Thr Cys Thr Asp Tyr Leu Gly Gly Phe Ser 1155 1160 1165tgc aag tgt gtg gct ggc tac cat ggg tct aac tgc tcc gag gag 3627Cys Lys Cys Val Ala Gly Tyr His Gly Ser Asn Cys Ser Glu Glu 1170 1175 1180atc aac gag tgc ctg tcc cag ccc tgc cag aat ggg ggt acc tgc 3672Ile Asn Glu Cys Leu Ser Gln Pro Cys Gln Asn Gly Gly Thr Cys 1185 1190 1195att gat ctg acc aac tcc tac aag tgt tcc tgc ccc cgg ggg aca 3717Ile Asp Leu Thr Asn Ser Tyr Lys Cys Ser Cys Pro Arg Gly Thr 1200 1205 1210cag ggt gta cac tgt gag atc aat gtt gat gac tgc cat ccc ccc 3762Gln Gly Val His Cys Glu Ile Asn Val Asp Asp Cys His Pro Pro 1215 1220 1225ctt gac cct gcc tcc cga agc ccc aag tgc ttc aac aat ggc acc 3807Leu Asp Pro Ala Ser Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr 1230 1235 1240tgt gtg gac cag gtg ggt ggc tat acc tgc acc tgc cca cca ggc 3852Cys Val Asp Gln Val Gly Gly Tyr Thr Cys Thr Cys Pro Pro Gly 1245 1250 1255ttc gtc ggg gag cgg tgt gag ggt gat gtc aat gaa tgt ctc tcc 3897Phe Val Gly Glu Arg Cys Glu Gly Asp Val Asn Glu Cys Leu Ser 1260 1265 1270aac ccc tgt gac cca cgt ggc acc cag aac tgt gtg cag cgt gtt 3942Asn Pro Cys Asp Pro Arg Gly Thr Gln Asn Cys Val Gln Arg Val 1275 1280 1285aat gac ttc cac tgc gag tgc cgg gct ggc cac act gga cgc cgc 3987Asn Asp Phe His Cys Glu Cys Arg Ala Gly His Thr Gly Arg Arg 1290 1295 1300tgt gag tca gtc atc aat ggc tgc agg ggc aaa cct tgc aag aat 4032Cys Glu Ser Val Ile Asn Gly Cys Arg Gly Lys Pro Cys Lys Asn 1305 1310 1315ggg ggt gtc tgt gcc gtg gcc tcc aac acc gcc cgt gga ttc atc 4077Gly Gly Val Cys Ala Val Ala Ser Asn Thr Ala Arg Gly Phe Ile 1320 1325 1330tgt agg tgc cct gcg ggc ttc gag ggt gcc aca tgt gag aat gat 4122Cys Arg Cys Pro Ala Gly Phe Glu Gly Ala Thr Cys Glu Asn Asp 1335 1340 1345gcc cgc act tgt ggc agc tta cgc tgt ctc aac ggt ggt aca tgc 4167Ala Arg Thr Cys Gly Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys 1350 1355 1360atc tcg ggc cca cgt agt ccc acc tgc cta tgc ctg gga tcc ttc 4212Ile Ser Gly Pro Arg Ser Pro Thr Cys Leu Cys Leu Gly Ser Phe 1365 1370 1375acc ggc cct gag tgc cag ttc cca gcc agc agc ccc tgt gtg ggt 4257Thr Gly Pro Glu Cys Gln Phe Pro Ala Ser Ser Pro Cys Val Gly 1380 1385 1390agc aac ccc tgc tac aat cag ggc acc tgt gag ccc aca tcc gag 4302Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu 1395 1400 1405aac cct ttc tac cgc tgt cta tgc cct gcc aaa ttc aac ggg cta 4347Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu 1410 1415 1420ctg tgc cac atc ctg gac tac agc ttc aca ggt ggc gct ggc ccg 4392Leu Cys His Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Pro 1425 1430 1435gac att ccc cca ccg cag att gag gag gcc tgt gag ctg cct gag 4437Asp Ile Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu 1440 1445 1450tgc cag gtg gat gca ggc aat aag gtc tgc aac ctg cag tgt aat 4482Cys Gln Val Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn 1455 1460 1465aat cac gca tgt ggc tgg gat ggt ggc gac tgc tcc ctc aac ttc 4527Asn His Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe 1470 1475 1480aat gac ccc tgg aag aac tgc acg cag tct cta cag tgc tgg aag 4572Asn Asp Pro Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys 1485 1490 1495tat ttt agc gac ggc cac tgt gac agc cag tgc aac tcg gcc ggc 4617Tyr Phe Ser Asp Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly 1500 1505 1510tgc ctc ttt gat ggc ttc gac tgc cag ctc acc gag gga cag tgc 4662Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys 1515 1520 1525aac ccc ctg tat gac cag tac tgc aag gac cac ttc agt gat ggc 4707Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His Phe Ser Asp Gly 1530 1535 1540cac tgc gac cag ggc tgt aac agt gcc gaa tgt gag tgg gat ggc 4752His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu Trp Asp Gly 1545 1550 1555cta gac tgt gct gag cat gta ccc gag cgg ctg gca gcg ggc acc 4797Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala Gly Thr 1560 1565 1570ctg gtc ctg gtg gtg ctg ctt cca ccc gac cag cta cgg aac aac 4842Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn 1575 1580 1585tcc ttc cac ttt ctg cgg gag ctc agc cac gtg ctg cac acc aac 4887Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn 1590 1595 1600gtg gtc ttc aag cgt gat gcg caa ggc cag cag atg atc ttc ccg 4932Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro 1605 1610 1615tac tat ggc cac gag gaa gag ctg cgc aag cac cca atc aag cgc 4977Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg 1620 1625 1630tct aca gtg ggt tgg gcc acc tct tca ctg ctt cct ggt acc agt 5022Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser 1635 1640 1645ggt ggg cgc cag cgc agg gag ctg gac ccc atg gac atc cgt ggc 5067Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly 1650 1655 1660tcc att gtc tac ctg gag atc gac aac cgg caa tgt gtg cag tca 5112Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser 1665 1670 1675tcc tcg cag tgc ttc cag agt gcc acc gat gtg gct gcc ttc cta 5157Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu 1680 1685 1690ggt gct ctt gcg tca ctt ggc agc ctc aat att cct tac aag att 5202Gly Ala Leu Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile 1695 1700 1705gag gcc gtg aag agt gag ccg gtg gag cct ccg ctg ccc tcg cag 5247Glu Ala Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln 1710 1715 1720ctg cac ctc atg tac gtg gca gcg gcc gcc ttc gtg ctc ctg ttc 5292Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe 1725 1730 1735ttt gtg ggc tgt ggg gtg ctg ctg tcc cgc aag cgc cgg cgc cag 5337Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln 1740 1745 1750cat ggc cag ctc tgg ttc cct gag ggt ttc aaa gtg tca gag gcc 5382His Gly Gln Leu Trp Phe Pro Glu Gly Phe Lys Val Ser Glu Ala 1755 1760 1765agc aag aag aag cgg aga gag ccc ctc ggc gag gac tca gtc ggc 5427Ser Lys Lys Lys Arg Arg Glu Pro Leu Gly Glu Asp Ser Val Gly 1770 1775 1780ctc aag ccc ctg aag aat gcc tca gat ggt gct ctg atg gac gac 5472Leu Lys Pro Leu Lys Asn Ala Ser Asp Gly Ala Leu Met Asp Asp 1785 1790 1795aat cag aac gag tgg gga gac gaa gac ctg gag acc aag aag ttc 5517Asn Gln Asn Glu Trp Gly Asp Glu Asp Leu Glu Thr Lys Lys Phe 1800 1805 1810cgg ttt gag gag cca gta gtt ctc cct gac ctg agt gat cag act 5562Arg Phe Glu Glu Pro Val Val Leu Pro Asp Leu Ser Asp Gln Thr 1815 1820 1825gac cac agg cag tgg acc cag cag cac ctg gac gct gct gac ctg 5607Asp His Arg Gln Trp Thr Gln Gln His Leu Asp Ala Ala Asp Leu 1830 1835 1840cgc atg tct gcc atg gcc cca aca ccg cct cag ggg gag gtg gat 5652Arg Met Ser Ala Met Ala Pro Thr Pro Pro Gln Gly Glu Val Asp 1845 1850 1855gct gac tgc atg gat gtc aat gtt cga gga cca gat ggc ttc aca 5697Ala Asp Cys Met Asp Val Asn Val Arg Gly Pro Asp Gly Phe Thr 1860 1865 1870ccc ctc atg att gcc tcc tgc agt gga ggg ggc ctt gag aca ggc 5742Pro Leu Met Ile Ala Ser Cys Ser Gly Gly Gly Leu Glu Thr Gly 1875 1880 1885aac agt gaa gaa gaa gaa gat gca cct gct gtc atc tct gac ttc 5787Asn Ser Glu Glu Glu Glu Asp Ala Pro Ala Val Ile Ser Asp Phe 1890 1895 1900atc tac cag ggc gcc agc ttg cac aac cag aca gac cgc acc ggg 5832Ile Tyr Gln Gly Ala Ser Leu His Asn Gln Thr Asp Arg Thr Gly 1905 1910 1915gag acc gcc ttg cac ttg gct gcc cga tac tct cgt tca gat cgt 5877Glu Thr Ala Leu His Leu Ala Ala Arg Tyr Ser Arg Ser Asp Arg 1920 1925 1930cga aag cgc ctt gag gcc agt gca gat gcc aac atc cag gac aac 5922Arg Lys Arg Leu Glu Ala Ser Ala Asp Ala Asn Ile Gln Asp Asn 1935 1940 1945atg ggc cgt act ccg tta cat gca gca gtt tct gca gat gct cag 5967Met Gly Arg Thr Pro Leu His Ala Ala Val Ser Ala Asp Ala Gln 1950 1955 1960ggt gtc ttc cag atc ctg ctc cgg aac agg gcc aca gat ctg gat 6012Gly Val Phe Gln Ile Leu Leu Arg Asn Arg Ala Thr Asp Leu Asp 1965 1970 1975gcc cga atg cat gat ggc aca act cca ctg atc ctg gct gcg cgc 6057Ala Arg Met His Asp Gly Thr Thr Pro Leu Ile Leu Ala Ala Arg 1980 1985 1990ctg gcc gtg gag ggc atg ctg gag gac ctc atc aac tca cat gct 6102Leu Ala Val Glu Gly Met Leu Glu Asp Leu Ile Asn Ser His Ala 1995 2000 2005gac gtc aat gcc gtg gat gac cta ggc aag tcg gct ttg cat tgg 6147Asp Val Asn Ala Val Asp Asp Leu Gly Lys Ser Ala Leu His Trp 2010 2015 2020gcg gcc gcg gtg aac aat gtg gat gct gct gtt gtg ctc ctg aag 6192Ala Ala Ala Val Asn Asn Val Asp Ala Ala Val Val Leu Leu Lys 2025 2030 2035aac gga gcc aac aag gac atc gag aac aac aag gag gag act tcc 6237Asn Gly Ala Asn Lys Asp Ile Glu Asn Asn Lys Glu Glu Thr Ser 2040 2045 2050ctg ttc ctg tcg atc cgc cgt gag agc tat gag act gcc aaa gtg 6282Leu Phe Leu Ser Ile Arg Arg Glu Ser Tyr Glu Thr Ala Lys Val 2055 2060 2065ttg ctg gac cac ttt gcc aac cgg gac atc acg gat cac atg gac 6327Leu Leu Asp His Phe Ala Asn Arg Asp Ile Thr Asp His Met Asp 2070 2075 2080cga ttg ccg cgg gac atc gca cag gag cgt atg cac cac gat atc 6372Arg Leu Pro Arg Asp Ile Ala Gln Glu Arg Met His His Asp Ile 2085 2090 2095gtg cgg ctt ttg gat gag tac aac ctg gtg cgg tcc cca cag ctg 6417Val Arg Leu Leu Asp Glu Tyr Asn Leu Val Arg Ser Pro Gln Leu 2100 2105 2110cat ggc act gcc ctg ggt ggc aca ccc act ctg tct ccc aca ctc 6462His Gly Thr Ala Leu Gly Gly Thr Pro Thr Leu Ser Pro Thr Leu 2115 2120 2125tgc tcg cca aat ggc tac cct ggc aat ctc aag tct gcc aca cag 6507Cys Ser Pro Asn Gly Tyr Pro Gly Asn Leu Lys Ser Ala Thr Gln 2130 2135 2140ggc aag aag gcc cgc aag cca agc acc aaa ggg ctg gct tgt ggt 6552Gly Lys Lys Ala Arg Lys Pro Ser Thr Lys Gly Leu Ala Cys Gly 2145 2150 2155agc aag gaa gct aag gac ctc aag gca cgg agg aag agt tcc cag 6597Ser Lys Glu Ala Lys Asp Leu Lys Ala Arg Arg Lys Ser Ser Gln 2160 2165 2170gat ggc aag ggc tgg ctg ttg gac agc tcg tcg agc atg ctg tcg 6642Asp Gly Lys Gly Trp Leu Leu Asp Ser Ser Ser Ser Met Leu Ser 2175 2180 2185cct gtg gac tcc ctc gag tca ccc cat ggc tac ttg tca gat gtg 6687Pro Val Asp Ser Leu Glu Ser Pro His Gly Tyr Leu Ser Asp Val 2190 2195 2200gcc tcg cac ccc ctc ctc ccc tcc cca ttc cag cag tct cca tcc 6732Ala Ser His Pro Leu Leu Pro Ser Pro Phe Gln Gln Ser Pro Ser 2205 2210 2215atg cct ctc agc cac ctg cct ggt atg cct gac acc cac ctg ggc 6777Met Pro Leu Ser His Leu Pro Gly Met Pro Asp Thr His Leu Gly 2220 2225 2230atc agc cac ttg aat gtg gca gcc aag cct gag atg gca gca ctg 6822Ile Ser His Leu Asn Val Ala Ala Lys Pro Glu Met Ala Ala Leu 2235 2240 2245gct gga ggt agc cgg ttg gcc ttt gag cac ccc ccg cca cgc ctc 6867Ala Gly Gly Ser Arg Leu Ala Phe Glu His Pro Pro Pro Arg Leu 2250 2255 2260tcc cac ctg cct gta gcc tcc agt gcc tgc aca gtg ctg agt acc
6912Ser His Leu Pro Val Ala Ser Ser Ala Cys Thr Val Leu Ser Thr 2265 2270 2275aat ggc acc ggg gct atg aat ttc acc gtg ggt gca ccg gca agc 6957Asn Gly Thr Gly Ala Met Asn Phe Thr Val Gly Ala Pro Ala Ser 2280 2285 2290ttg aat ggc cag tgt gag tgg ctt ccc cgg ctc cag aat ggc atg 7002Leu Asn Gly Gln Cys Glu Trp Leu Pro Arg Leu Gln Asn Gly Met 2295 2300 2305gtg ccc agc cag tac aac cca cta cgg ccg ggt gtg acg ccg ggc 7047Val Pro Ser Gln Tyr Asn Pro Leu Arg Pro Gly Val Thr Pro Gly 2310 2315 2320aca ctg agc aca cag gca gct ggg ctc cag cat agc atg atg ggg 7092Thr Leu Ser Thr Gln Ala Ala Gly Leu Gln His Ser Met Met Gly 2325 2330 2335cca cta cac agc agc ctc tcc acc aat acc ttg tcc ccg att att 7137Pro Leu His Ser Ser Leu Ser Thr Asn Thr Leu Ser Pro Ile Ile 2340 2345 2350tac cag ggc ctg ccc aac aca cgg ctg gca aca cag cct cac ctg 7182Tyr Gln Gly Leu Pro Asn Thr Arg Leu Ala Thr Gln Pro His Leu 2355 2360 2365gtg cag acc cag cag gtg cag cca cag aac tta cca ctc cag cca 7227Val Gln Thr Gln Gln Val Gln Pro Gln Asn Leu Pro Leu Gln Pro 2370 2375 2380cag aac tta cag cca cca tca cag cca cac ctc agt gtg agc tcg 7272Gln Asn Leu Gln Pro Pro Ser Gln Pro His Leu Ser Val Ser Ser 2385 2390 2395gca gcc aat ggg cac ctg ggg cgg agc ttc ttg agt ggg gag ccc 7317Ala Ala Asn Gly His Leu Gly Arg Ser Phe Leu Ser Gly Glu Pro 2400 2405 2410agt cag gca gat gta caa ccg ctg ggc ccc agc agt ctg cct gtg 7362Ser Gln Ala Asp Val Gln Pro Leu Gly Pro Ser Ser Leu Pro Val 2415 2420 2425cac acc att ctg ccc cag gaa agc cag gcc cta cca aca tca ctg 7407His Thr Ile Leu Pro Gln Glu Ser Gln Ala Leu Pro Thr Ser Leu 2430 2435 2440cca tcc tcc atg gtc cca ccc atg acc act acc cag ttc ctg acc 7452Pro Ser Ser Met Val Pro Pro Met Thr Thr Thr Gln Phe Leu Thr 2445 2450 2455cct cca tca cag cac agt tac tcc tcc tcc cct gtg gac aac acc 7497Pro Pro Ser Gln His Ser Tyr Ser Ser Ser Pro Val Asp Asn Thr 2460 2465 2470ccc agc cac cag ctg cag gtg cca gag ccc act ttc ctc acc cca 7542Pro Ser His Gln Leu Gln Val Pro Glu Pro Thr Phe Leu Thr Pro 2475 2480 2485tcc cct gag tcc cct gac cag tgg tcc agc tcc tcc ccg cat tcc 7587Ser Pro Glu Ser Pro Asp Gln Trp Ser Ser Ser Ser Pro His Ser 2490 2495 2500aac atc tct gat tgg tcc gag ggc atc tcc agc ccg ccc acc acc 7632Asn Ile Ser Asp Trp Ser Glu Gly Ile Ser Ser Pro Pro Thr Thr 2505 2510 2515atg ccg tcc cag atc acc cac att cca gag gca ttt aaa taaacagaga 7681Met Pro Ser Gln Ile Thr His Ile Pro Glu Ala Phe Lys 2520 2525 2530tgtgggatgc aggaccccag cttccgttcc caagccctgt tggaagtcct ttccagtgct 7741tcaggatgct ggggcgacca aaggagcttt ttaaaaaatg tttttataca aaataagagg 7801acaagaattt catttttttt tttagtattt atttatgtac ttttattttc cacagaaaca 7861ctgccttttt atttatatgt attgttttct atggcactag gggaaaaaca tatctgttcc 7921aagaaaataa actagttctc agagccttga ttttcctggt cagggtgaag ttccctgtgt 7981gtctgtaaaa tatgaacaag gattcatgat ttgtaaatgc tgtttattta ttgattgctt 8041ctttccaaaa tcgaaaaaaa aaa 806443641DNAHomo sapiensCDS(195)..(2504) 4gctgactcgc ctggctctga gccccgccgc cgcgctcggg ctccgtcagt ttcctcggca 60gcggtaggcg agagcacgcg gaggagcgtg cgcgggggcc ccgggagacg gcggcggtgg 120cggcgcgggc agagcaagga cgcggcggat cccactcgca cagcagcgca ctcggtgccc 180cgcgcagggt cgcg atg ctg ccc ggt ttg gca ctg ctc ctg ctg gcc gcc 230Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala1 5 10tgg acg gct cgg gcg ctg gag gta ccc act gat ggt aat gct ggc ctg 278Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu 15 20 25ctg gct gaa ccc cag att gcc atg ttc tgt ggc aga ctg aac atg cac 326Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His 30 35 40atg aat gtc cag aat ggg aag tgg gat tca gat cca tca ggg acc aaa 374Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys 45 50 55 60acc tgc att gat acc aag gaa ggc atc ctg cag tat tgc caa gaa gtc 422Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val65 70 75tac cct gaa ctg cag atc acc aat gtg gta gaa gcc aac caa cca gtg 470Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val 80 85 90acc atc cag aac tgg tgc aag cgg ggc cgc aag cag tgc aag acc cat 518Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His 95 100 105ccc cac ttt gtg att ccc tac cgc tgc tta gtt ggt gag ttt gta agt 566Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser 110 115 120gat gcc ctt ctc gtt cct gac aag tgc aaa ttc tta cac cag gag agg 614Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg 125 130 135 140atg gat gtt tgc gaa act cat ctt cac tgg cac acc gtc gcc aaa gag 662Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu145 150 155aca tgc agt gag aag agt acc aac ttg cat gac tac ggc atg ttg ctg 710Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu 160 165 170ccc tgc gga att gac aag ttc cga ggg gta gag ttt gtg tgt tgc cca 758Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro 175 180 185ctg gct gaa gaa agt gac aat gtg gat tct gct gat gcg gag gag gat 806Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp 190 195 200gac tcg gat gtc tgg tgg ggc gga gca gac aca gac tat gca gat ggg 854Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly 205 210 215 220agt gaa gac aaa gta gta gaa gta gca gag gag gaa gaa gtg gct gag 902Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu225 230 235gtg gaa gaa gaa gaa gcc gat gat gac gag gac gat gag gat ggt gat 950Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp 240 245 250gag gta gag gaa gag gct gag gaa ccc tac gaa gaa gcc aca gag aga 998Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg 255 260 265acc acc agc att gcc acc acc acc acc acc acc aca gag tct gtg gaa 1046Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu 270 275 280gag gtg gtt cga gag gtg tgc tct gaa caa gcc gag acg ggg ccg tgc 1094Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys 285 290 295 300cga gca atg atc tcc cgc tgg tac ttt gat gtg act gaa ggg aag tgt 1142Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys305 310 315gcc cca ttc ttt tac ggc gga tgt ggc ggc aac cgg aac aac ttt gac 1190Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp 320 325 330aca gaa gag tac tgc atg gcc gtg tgt ggc agc gcc atg tcc caa agt 1238Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser 335 340 345tta ctc aag act acc cag gaa cct ctt gcc cga gat cct gtt aaa ctt 1286Leu Leu Lys Thr Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu 350 355 360cct aca aca gca gcc agt acc cct gat gcc gtt gac aag tat ctc gag 1334Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu 365 370 375 380aca cct ggg gat gag aat gaa cat gcc cat ttc cag aaa gcc aaa gag 1382Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu385 390 395agg ctt gag gcc aag cac cga gag aga atg tcc cag gtc atg aga gaa 1430Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu 400 405 410tgg gaa gag gca gaa cgt caa gca aag aac ttg cct aaa gct gat aag 1478Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys 415 420 425aag gca gtt atc cag cat ttc cag gag aaa gtg gaa tct ttg gaa cag 1526Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln 430 435 440gaa gca gcc aac gag aga cag cag ctg gtg gag aca cac atg gcc aga 1574Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg 445 450 455 460gtg gaa gcc atg ctc aat gac cgc cgc cgc ctg gcc ctg gag aac tac 1622Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr465 470 475atc acc gct ctg cag gct gtt cct cct cgg cct cgt cac gtg ttc aat 1670Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn 480 485 490atg cta aag aag tat gtc cgc gca gaa cag aag gac aga cag cac acc 1718Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr 495 500 505cta aag cat ttc gag cat gtg cgc atg gtg gat ccc aag aaa gcc gct 1766Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala 510 515 520cag atc cgg tcc cag gtt atg aca cac ctc cgt gtg att tat gag cgc 1814Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg 525 530 535 540atg aat cag tct ctc tcc ctg ctc tac aac gtg cct gca gtg gcc gag 1862Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu545 550 555gag att cag gat gaa gtt gat gag ctg ctt cag aaa gag caa aac tat 1910Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr 560 565 570tca gat gac gtc ttg gcc aac atg att agt gaa cca agg atc agt tac 1958Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr 575 580 585gga aac gat gct ctc atg cca tct ttg acc gaa acg aaa acc acc gtg 2006Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val 590 595 600gag ctc ctt ccc gtg aat gga gag ttc agc ctg gac gat ctc cag ccg 2054Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro 605 610 615 620tgg cat tct ttt ggg gct gac tct gtg cca gcc aac aca gaa aac gaa 2102Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu625 630 635gtt gag cct gtt gat gcc cgc cct gct gcc gac cga gga ctg acc act 2150Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr 640 645 650cga cca ggt tct ggg ttg aca aat atc aag acg gag gag atc tct gaa 2198Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu 655 660 665gtg aag atg gat gca gaa ttc cga cat gac tca gga tat gaa gtt cat 2246Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His 670 675 680cat caa aaa ttg gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt 2294His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly 685 690 695 700gca atc att gga ctc atg gtg ggc ggt gtt gtc ata gcg aca gtg atc 2342Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile705 710 715gtc atc acc ttg gtg atg ctg aag aag aaa cag tac aca tcc att cat 2390Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His 720 725 730cat ggt gtg gtg gag gtt gac gcc gct gtc acc cca gag gag cgc cac 2438His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His 735 740 745ctg tcc aag atg cag cag aac ggc tac gaa aat cca acc tac aag ttc 2486Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe 750 755 760ttt gag cag atg cag aac tagacccccg ccacagcagc ctctgaagtt 2534Phe Glu Gln Met Gln Asn 765 770ggacagcaaa accattgctt cactacccat cggtgtccat ttatagaata atgtgggaag 2594aaacaaaccc gttttatgat ttactcatta tcgccttttg acagctgtgc tgtaacacaa 2654gtagatgcct gaacttgaat taatccacac atcagtaatg tattctatct ctctttacat 2714tttggtctct atactacatt attaatgggt tttgtgtact gtaaagaatt tagctgtatc 2774aaactagtgc atgaatagat tctctcctga ttatttatca catagcccct tagccagttg 2834tatattattc ttgtggtttg tgacccaatt aagtcctact ttacatatgc tttaagaatc 2894gatgggggat gcttcatgtg aacgtgggag ttcagctgct tctcttgcct aagtattcct 2954ttcctgatca ctatgcattt taaagttaaa catttttaag tatttcagat gctttagaga 3014gatttttttt ccatgactgc attttactgt acagattgct gcttctgcta tatttgtgat 3074ataggaatta agaggataca cacgtttgtt tcttcgtgcc tgttttatgt gcacacatta 3134ggcattgaga cttcaagctt ttcttttttt gtccacgtat ctttgggtct ttgataaaga 3194aaagaatccc tgttcattgt aagcactttt acggggcggg tggggagggg tgctctgctg 3254gtcttcaatt accaagaatt ctccaaaaca attttctgca ggatgattgt acagaatcat 3314tgcttatgac atgatcgctt tctacactgt attacataaa taaattaaat aaaataaccc 3374cgggcaagac ttttctttga aggatgacta cagacattaa ataatcgaag taattttggg 3434tggggagaag aggcagattc aattttcttt aaccagtctg aagtttcatt tatgatacaa 3494aagaagatga aaatggaagt ggcaatataa ggggatgagg aaggcatgcc tggacaaacc 3554cttcttttaa gatgtgtctt caatttgtat aaaatggtgt tttcatgtaa ataaatacat 3614tcttggagga gcaaaaaaaa aaaaaaa 3641522PRTMus musculus 5His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val1 5 10 15Gly Cys Gly Val Leu Leu 20666DNAMus musculusCDS(1)..(66) 6cac ctc atg tac gtg gca gcg gcc gcc ttc gtg ctc ctg ttc ttt gtg 48His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val1 5 10 15ggc tgt ggg gtg ctg ctg 66Gly Cys Gly Val Leu Leu 20724PRTHomo sapiens 7Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val1 5 10 15Ile Val Ile Thr Leu Val Met Leu 20872DNAHomo sapiensCDS(1)..(72) 8ggt gca atc att gga ctc atg gtg ggc ggt gtt gtc ata gcg aca gtg 48Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val1 5 10 15atc gtc atc acc ttg gtg atg ctg 72Ile Val Ile Thr Leu Val Met Leu 20999PRTMus musculus 9Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu1 5 10 15Val Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys 20 25 30Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln His Gly Gln Leu Trp 35 40 45Phe Pro Glu Gly Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg Arg 50 55 60Glu Pro Leu Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys Asn Ala65 70 75 80Ser Asp Gly Ala Leu Met Asp Asp Asn Gln Asn Glu Trp Gly Asp Glu 85 90 95Asp Leu Glu10297DNAMus musculusCDS(1)..(297) 10gtg aag agt gag ccg gtg gag cct ccg ctg ccc tcg cag ctg cac ctc 48Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu1 5 10 15gtg tac gtg gca gcg gcc gcc ttc gtg ctc ctg ttc ttt gtg ggc tgt 96Val Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys 20 25 30ggg gtg ctg ctg tcc cgc aag cgc cgg cgg cag cat ggc cag ctc tgg 144Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln His Gly Gln Leu Trp 35 40 45ttc cct gag ggt ttc aaa gtg tca gag gcc agc aag aag aag cgg aga 192Phe Pro Glu Gly Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg Arg 50 55 60gag ccc ctc ggc gag gac tca gtc ggc ctc aag ccc ctg aag aat gcc 240Glu Pro Leu Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys Asn Ala65 70 75 80tca gat ggt gct ctg atg gac gac aat cag aac gag tgg gga gac gaa 288Ser Asp Gly Ala Leu Met Asp Asp Asn Gln Asn Glu Trp Gly Asp Glu 85 90 95gac ctg gag 297Asp Leu Glu 1199PRTHomo sapiens 11 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr 35 40 45Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val 50 55 60Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys65 70 75 80Met Gln Gln Asn Gly
Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln 85 90 95Met Gln Asn12297DNAHomo sapiensCDS(1)..(297) 12gat gca gaa ttc cga cat gac tca gga tat gaa gtt cat cat caa aaa 48Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15ttg gtg ttc ttt gca gaa gat gtg ggt tca aac aaa ggt gca atc att 96Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30gga ctc atg gtg ggc ggt gtt gtc ata gcg aca gtg atc gtc atc acc 144Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr 35 40 45ttg gtg atg ctg aag aag aaa cag tac aca tcc att cat cat ggt gtg 192Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val 50 55 60gtg gag gtt gac gcc gct gtc acc cca gag gag cgc cac ctg tcc aag 240Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys65 70 75 80atg cag cag aac ggc tac gaa aat cca acc tac aag ttc ttt gag cag 288Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln 85 90 95atg cag aac 297Met Gln Asn 13351DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 13atg gtg aag agt gag ccg gtg gag cct ccg ctg ccc tcg cag ctg cac 48Met Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His1 5 10 15ctc gtg tac gtg gca gcg gcc gcc ttc gtg ctc ctg ttc ttt gtg ggc 96Leu Val Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 20 25 30tgt ggg gtg ctg ctg tcc cgc aag cgc cgg cgg cag cat ggc cag ctc 144Cys Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln His Gly Gln Leu 35 40 45tgg ttc cct gag ggt ttc aaa gtg tca gag gcc agc aag aag aag cgg 192Trp Phe Pro Glu Gly Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg 50 55 60aga gag ccc ctc ggc gag gac tca gtc ggc ctc aag ccc ctg aag aat 240Arg Glu Pro Leu Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys Asn65 70 75 80gcc tca gat ggt gct ctg atg gac gac aat cag aac gag tgg gga gac 288Ala Ser Asp Gly Ala Leu Met Asp Asp Asn Gln Asn Glu Trp Gly Asp 85 90 95gaa gac ctg gag gac tac aag gac gat gac gat aag gga tcc cat cac 336Glu Asp Leu Glu Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser His His 100 105 110cat cac cat cac tag 351His His His His 11514116PRTArtificial SequenceDescription of Artificial Sequence Synthetic primer 14Met Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His1 5 10 15Leu Val Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 20 25 30Cys Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln His Gly Gln Leu 35 40 45Trp Phe Pro Glu Gly Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg 50 55 60Arg Glu Pro Leu Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys Asn65 70 75 80Ala Ser Asp Gly Ala Leu Met Asp Asp Asn Gln Asn Glu Trp Gly Asp 85 90 95Glu Asp Leu Glu Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser His His 100 105 110His His His His 11515100PRTArtificial SequenceDescription of Artificial Sequence Synthetic primer 15Met Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His1 5 10 15Leu Val Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 20 25 30Cys Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln His Gly Gln Leu 35 40 45Trp Phe Pro Glu Gly Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg 50 55 60Arg Glu Pro Leu Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys Asn65 70 75 80Ala Ser Asp Gly Ala Leu Met Asp Asp Asn Gln Asn Glu Trp Gly Asp 85 90 95Glu Asp Leu Glu 1001616PRTArtificial SequenceDescription of Artificial Sequence Synthetic primer 16Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser His His His His His His1 5 10 15172531PRTMus musculus 17Met Pro Arg Leu Leu Thr Pro Leu Leu Cys Leu Thr Leu Leu Pro Ala1 5 10 15Arg Ala Ala Arg Gly Leu Arg Cys Ser Gln Pro Ser Gly Thr Cys Leu 20 25 30Asn Gly Gly Arg Cys Glu Val Ala Ser Gly Thr Glu Ala Cys Val Ala 35 40 45Ser Gly Ser Phe Val Gly Gln Arg Cys Gln Asp Pro Asn Pro Cys Leu 50 55 60Ser Thr Arg Cys Lys Asn Ala Gly Thr Cys Tyr Val Val Asp His Gly65 70 75 80Gly Ile Val Asp Tyr Ala Cys Ser Cys Pro Leu Gly Phe Ser Gly Pro 85 90 95Leu Cys Leu Thr Pro Leu Asp Lys Pro Cys Leu Ala Asn Pro Cys Arg 100 105 110Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys Arg 115 120 125Cys Ser Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 130 135 140Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ser145 150 155 160 Ser Tyr Ile Cys Arg Cys Pro Pro Gly Phe His Gly Pro Thr Cys Arg 165 170 175 Gln Asp Val Asn Glu Cys Ser Gln Asn Pro Gly Leu Cys Arg His Gly 180 185 190 Gly His Cys His Asn Glu Ile Gly Ser Tyr Arg Cys Ala Cys Cys Ala 195 200 205 Thr His Thr Gly Pro His Cys Glu Leu Pro Tyr Val Pro Cys Ser Pro 210 215 220Ser Pro Cys Gln Asn Gly Ala Thr Cys Arg Pro Thr Gly Asp Thr Thr225 230 235 240His Glu Cys Ala Cys Leu Pro Gly Phe Ala Gly Gln Asn Cys Glu Glu 245 250 255Asn Val Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 260 265 270Val Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro Glu Val Thr 275 280 285Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Met Pro Asn 290 295 300Ala Cys Gln Asn Ala Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn305 310 315 320Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile 325 330 335Asp Asp Cys Ala Ser Ala Ala Cys Phe Gln Gly Ala Thr Cys His Asp 340 345 350Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr Gly Leu 355 360 365Leu Cys His Leu Lys His Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly 370 375 380Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Arg Ile Cys Thr Cys385 390 395 400Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys 405 410 415Asp Leu Gly Ala Asn Arg Cys Glu His Ala Gly Lys Cys Leu Asn Thr 420 425 430Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Gly 435 440 445Cys Glu Ile Asp Val Asn Glu Cys Ile Ser Asn Pro Cys Gln Asn Asp 450 455 460Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys Ile Cys Met Pro465 470 475 480Gly Tyr Glu Gly Val Tyr Cys Glu Ile Asn Thr Asp Glu Cys Ala Ser 485 490 495Ser Pro Cys Leu His Asn Gly His Cys Met Asp Lys Ile His Glu Phe 500 505 510Gln Cys Gln Cys Pro Lys Gly Phe Asn Gly His Leu Cys Gln Tyr Asp 515 520 525Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu 530 535 540Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly545 550 555 560Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His 565 570 575Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Gln 580 585 590Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys His 595 600 605Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln Asp Arg Asp Asn Ser 610 615 620Tyr Leu Cys Leu Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile625 630 635 640Asn Leu Asp Asp Cys Ala Ser Asn Pro Cys Asp Ser Gly Thr Cys Leu 645 650 655Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly 660 665 670Ser Met Cys Asn Val Asn Ile Asp Glu Cys Ala Gly Ser Pro Cys His 675 680 685Asn Gly Gly Thr Cys Glu Asp Gly Ile Ala Gly Phe Thr Cys Arg Cys 690 695 700Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys705 710 715 720Asn Ser Asn Pro Cys Ile His Gly Ala Cys Arg Asp Gly Leu Asn Gly 725 730 735Tyr Lys Cys Asp Cys Ala Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile 740 745 750Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 755 760 765Lys Asp Met Thr Ser Gly Tyr Val Cys Thr Cys Arg Glu Gly Phe Ser 770 775 780Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys785 790 795 800Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn 805 810 815Cys Pro Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 820 825 830Cys Ala Thr Ser Pro Cys Lys Asn Ser Gly Val Cys Lys Glu Ser Glu 835 840 845Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly Trp Gln Gly Gln 850 855 860Thr Cys Glu Val Asp Ile Asn Glu Cys Val Lys Ser Pro Cys Arg His865 870 875 880Gly Ala Ser Cys Gln Asn Thr Asn Gly Ser Tyr Arg Cys Leu Cys Gln 885 890 895Ala Gly Tyr Thr Gly Arg Asn Cys Glu Ser Asp Ile Asp Asp Cys Arg 900 905 910Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn Thr 915 920 925Ala Phe Cys Asp Cys Leu Pro Gly Phe Gln Gly Ala Phe Cys Glu Glu 930 935 940Asp Ile Asn Glu Cys Ala Ser Asn Pro Cys Gln Asn Gly Ala Asn Cys945 950 955 960Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Val Gly Phe Asn 965 970 975Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser Cys 980 985 990Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys Leu 995 1000 1005Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln Tyr Asp Val Asn 1010 1015 1020Glu Cys Asp Ser Arg Pro Cys Leu His Gly Gly Thr Cys Gln Asp 1025 1030 1035Ser Tyr Gly Thr Tyr Lys Cys Thr Cys Pro Gln Gly Tyr Thr Gly 1040 1045 1050Leu Asn Cys Gln Asn Leu Val Arg Trp Cys Asp Ser Ala Pro Cys 1055 1060 1065Lys Asn Gly Gly Arg Cys Trp Gln Thr Asn Thr Gln Tyr His Cys 1070 1075 1080Glu Cys Arg Ser Gly Trp Thr Gly Val Asn Cys Asp Val Leu Ser 1085 1090 1095Val Ser Cys Glu Val Ala Ala Gln Lys Arg Gly Ile Asp Val Thr 1100 1105 1110Leu Leu Cys Gln His Gly Gly Leu Cys Val Asp Glu Gly Asp Lys 1115 1120 1125His Tyr Cys His Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys Glu 1130 1135 1140Asp Glu Val Asp Glu Cys Ser Pro Asn Pro Cys Gln Asn Gly Ala 1145 1150 1155Thr Cys Thr Asp Tyr Leu Gly Gly Phe Ser Cys Lys Cys Val Ala 1160 1165 1170Gly Tyr His Gly Ser Asn Cys Ser Glu Glu Ile Asn Glu Cys Leu 1175 1180 1185Ser Gln Pro Cys Gln Asn Gly Gly Thr Cys Ile Asp Leu Thr Asn 1190 1195 1200Ser Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly Val His Cys 1205 1210 1215Glu Ile Asn Val Asp Asp Cys His Pro Pro Leu Asp Pro Ala Ser 1220 1225 1230Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln Val 1235 1240 1245Gly Gly Tyr Thr Cys Thr Cys Pro Pro Gly Phe Val Gly Glu Arg 1250 1255 1260Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Pro 1265 1270 1275Arg Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys 1280 1285 1290Glu Cys Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val Ile 1295 1300 1305Asn Gly Cys Arg Gly Lys Pro Cys Lys Asn Gly Gly Val Cys Ala 1310 1315 1320Val Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys Arg Cys Pro Ala 1325 1330 1335Gly Phe Glu Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly 1340 1345 1350Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg 1355 1360 1365Ser Pro Thr Cys Leu Cys Leu Gly Ser Phe Thr Gly Pro Glu Cys 1370 1375 1380Gln Phe Pro Ala Ser Ser Pro Cys Val Gly Ser Asn Pro Cys Tyr 1385 1390 1395Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu Asn Pro Phe Tyr Arg 1400 1405 1410Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu Cys His Ile Leu 1415 1420 1425Asp Tyr Ser Phe Thr Gly Gly Ala Gly Pro Asp Ile Pro Pro Pro 1430 1435 1440Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala 1445 1450 1455Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly 1460 1465 1470Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys 1475 1480 1485Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly 1490 1495 1500His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly 1505 1510 1515Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp 1520 1525 1530Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly 1535 1540 1545Cys Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu 1550 1555 1560His Val Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val 1565 1570 1575Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu 1580 1585 1590Arg Glu Leu Ser His Val Leu His Thr Asn Val Val Phe Lys Arg 1595 1600 1605Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr Tyr Gly His Glu 1610 1615 1620Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr Val Gly Trp 1625 1630 1635Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg Gln Arg 1640 1645 1650Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr Leu 1655 1660 1665Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe 1670 1675 1680Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser 1685 1690 1695Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser 1700 1705 1710Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr 1715 1720 1725Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly 1730 1735 1740Val Leu Leu Ser Arg Lys Arg Arg Arg Gln His Gly Gln Leu Trp 1745 1750 1755Phe Pro Glu Gly Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg 1760 1765 1770Arg Glu Pro Leu Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys 1775 1780 1785Asn Ala Ser Asp Gly Ala Leu Met Asp Asp Asn
Gln Asn Glu Trp 1790 1795 1800Gly Asp Glu Asp Leu Glu Thr Lys Lys Phe Arg Phe Glu Glu Pro 1805 1810 1815Val Val Leu Pro Asp Leu Ser Asp Gln Thr Asp His Arg Gln Trp 1820 1825 1830Thr Gln Gln His Leu Asp Ala Ala Asp Leu Arg Met Ser Ala Met 1835 1840 1845Ala Pro Thr Pro Pro Gln Gly Glu Val Asp Ala Asp Cys Met Asp 1850 1855 1860Val Asn Val Arg Gly Pro Asp Gly Phe Thr Pro Leu Met Ile Ala 1865 1870 1875Ser Cys Ser Gly Gly Gly Leu Glu Thr Gly Asn Ser Glu Glu Glu 1880 1885 1890Glu Asp Ala Pro Ala Val Ile Ser Asp Phe Ile Tyr Gln Gly Ala 1895 1900 1905Ser Leu His Asn Gln Thr Asp Arg Thr Gly Glu Thr Ala Leu His 1910 1915 1920Leu Ala Ala Arg Tyr Ser Arg Ser Asp Arg Arg Lys Arg Leu Glu 1925 1930 1935Ala Ser Ala Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr Pro 1940 1945 1950Leu His Ala Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile 1955 1960 1965Leu Leu Arg Asn Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp 1970 1975 1980Gly Thr Thr Pro Leu Ile Leu Ala Ala Arg Leu Ala Val Glu Gly 1985 1990 1995Met Leu Glu Asp Leu Ile Asn Ser His Ala Asp Val Asn Ala Val 2000 2005 2010Asp Asp Leu Gly Lys Ser Ala Leu His Trp Ala Ala Ala Val Asn 2015 2020 2025Asn Val Asp Ala Ala Val Val Leu Leu Lys Asn Gly Ala Asn Lys 2030 2035 2040Asp Ile Glu Asn Asn Lys Glu Glu Thr Ser Leu Phe Leu Ser Ile 2045 2050 2055Arg Arg Glu Ser Tyr Glu Thr Ala Lys Val Leu Leu Asp His Phe 2060 2065 2070Ala Asn Arg Asp Ile Thr Asp His Met Asp Arg Leu Pro Arg Asp 2075 2080 2085Ile Ala Gln Glu Arg Met His His Asp Ile Val Arg Leu Leu Asp 2090 2095 2100Glu Tyr Asn Leu Val Arg Ser Pro Gln Leu His Gly Thr Ala Leu 2105 2110 2115Gly Gly Thr Pro Thr Leu Ser Pro Thr Leu Cys Ser Pro Asn Gly 2120 2125 2130Tyr Pro Gly Asn Leu Lys Ser Ala Thr Gln Gly Lys Lys Ala Arg 2135 2140 2145Lys Pro Ser Thr Lys Gly Leu Ala Cys Gly Ser Lys Glu Ala Lys 2150 2155 2160Asp Leu Lys Ala Arg Arg Lys Ser Ser Gln Asp Gly Lys Gly Trp 2165 2170 2175Leu Leu Asp Ser Ser Ser Ser Met Leu Ser Pro Val Asp Ser Leu 2180 2185 2190Glu Ser Pro His Gly Tyr Leu Ser Asp Val Ala Ser His Pro Leu 2195 2200 2205Leu Pro Ser Pro Phe Gln Gln Ser Pro Ser Met Pro Leu Ser His 2210 2215 2220Leu Pro Gly Met Pro Asp Thr His Leu Gly Ile Ser His Leu Asn 2225 2230 2235Val Ala Ala Lys Pro Glu Met Ala Ala Leu Ala Gly Gly Ser Arg 2240 2245 2250Leu Ala Phe Glu His Pro Pro Pro Arg Leu Ser His Leu Pro Val 2255 2260 2265Ala Ser Ser Ala Cys Thr Val Leu Ser Thr Asn Gly Thr Gly Ala 2270 2275 2280Met Asn Phe Thr Val Gly Ala Pro Ala Ser Leu Asn Gly Gln Cys 2285 2290 2295Glu Trp Leu Pro Arg Leu Gln Asn Gly Met Val Pro Ser Gln Tyr 2300 2305 2310Asn Pro Leu Arg Pro Gly Val Thr Pro Gly Thr Leu Ser Thr Gln 2315 2320 2325Ala Ala Gly Leu Gln His Ser Met Met Gly Pro Leu His Ser Ser 2330 2335 2340Leu Ser Thr Asn Thr Leu Ser Pro Ile Ile Tyr Gln Gly Leu Pro 2345 2350 2355Asn Thr Arg Leu Ala Thr Gln Pro His Leu Val Gln Thr Gln Gln 2360 2365 2370Val Gln Pro Gln Asn Leu Pro Leu Gln Pro Gln Asn Leu Gln Pro 2375 2380 2385Pro Ser Gln Pro His Leu Ser Val Ser Ser Ala Ala Asn Gly His 2390 2395 2400Leu Gly Arg Ser Phe Leu Ser Gly Glu Pro Ser Gln Ala Asp Val 2405 2410 2415Gln Pro Leu Gly Pro Ser Ser Leu Pro Val His Thr Ile Leu Pro 2420 2425 2430Gln Glu Ser Gln Ala Leu Pro Thr Ser Leu Pro Ser Ser Met Val 2435 2440 2445Pro Pro Met Thr Thr Thr Gln Phe Leu Thr Pro Pro Ser Gln His 2450 2455 2460Ser Tyr Ser Ser Ser Pro Val Asp Asn Thr Pro Ser His Gln Leu 2465 2470 2475Gln Val Pro Glu Pro Thr Phe Leu Thr Pro Ser Pro Glu Ser Pro 2480 2485 2490Asp Gln Trp Ser Ser Ser Ser Pro His Ser Asn Ile Ser Asp Trp 2495 2500 2505Ser Glu Gly Ile Ser Ser Pro Pro Thr Thr Met Pro Ser Gln Ile 2510 2515 2520Thr His Ile Pro Glu Ala Phe Lys 2525 253018770PRTHomo sapiens 18Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg1 5 10 15Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu65 70 75 80Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu145 150 155 160Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu225 230 235 240Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe305 310 315 320Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340 345 350Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala385 390 395 400Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala 405 410 415Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu465 470 475 480Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe 500 505 510Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser 515 520 525Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser 530 535 540Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp545 550 555 560Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610 615 620Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val625 630 635 640Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu 675 680 685Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu705 710 715 720Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725 730 735Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met 755 760 765Gln Asn 770195PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 19Asp Tyr Ile Gly Ser1 5
Patent applications by Hiroko Iwanari, Tochigi JP
Patent applications by Masao Ohkuchi, Saitama JP
Patent applications by Takeshi Iwatsubo, Tokyo JP
Patent applications by Tatsuhiko Kodama, Tokyo JP
Patent applications by Yasuomi Urano, Tokyo JP
Patent applications by Perseus Proteomics Inc.
Patent applications by THE UNIVERSITY OF TOKYO
Patent applications in class Binds hormone or other secreted growth regulatory factor, differentiation factor, or intercellular mediator (e.g., cytokine, vascular permeability factor, etc.); or binds serum protein, plasma protein, fibrin, or enzyme
Patent applications in all subclasses Binds hormone or other secreted growth regulatory factor, differentiation factor, or intercellular mediator (e.g., cytokine, vascular permeability factor, etc.); or binds serum protein, plasma protein, fibrin, or enzyme